Curing for recursive nucleic acid-guided cell editing

ABSTRACT

The present disclosure provides automated multi-module instrumentation and automated methods for performing recursive editing of live cells with curing of editing vectors from prior rounds of editing.

RELATED CASES

The present application is a continuation of U.S. Ser. No. 16/892,679,entitled “Curing for Recursive Nucleic Acid-Guided Cell Editing”, filed4 Jun. 2020, which claims priority to U.S. Ser. No. 62/857,967, entitled“Curing for Recursive Nucleic Acid-Guided Cell Editing”, filed 6 Jun.2019.

FIELD OF THE INVENTION

The present disclosure relates to automated multi-module instruments,compositions and methods for performing recursive genomic editingtechnologies.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowfor manipulation of gene sequences, and hence gene function. Thenucleases include nucleic acid-guided nucleases and nuclease fusions,which enable researchers to generate permanent edits in live cells. Itis desirable to be able to perform two to many rounds of nucleicacid-guided nuclease editing sequentially (e.g., perform recursiveediting), but in doing so it is also desirable to clear or “cure” aprior editing nucleic acid from the cells before transforming ortransfecting the cells with a subsequent editing nucleic acid. Curing isa way to eliminate the prior editing vector including the attendant gRNAand donor DNA sequences editing or CREATE cassette) contained on anediting vector and also selection genes and other sequences contained onthe editing vector. Further, eliminating the editing vector from a priorround of editing permits a new editing vector to propagate within a cellwithout competition from the prior editing vector.

There is thus a need in the art of nucleic acid-guided nuclease geneediting for improved methods, compositions, modules and instruments forcuring editing vectors used in prior rounds of editing. The presentinvention satisfies this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides compositions, automated methods andmulti-module automated instrumentation for performing curing of editingvectors in recursive editing protocols.

Thus, in some embodiments there is provided a method for curing cellsduring recursive nucleic acid-directed nuclease editing comprising:designing and synthesizing sets of editing cassettes, wherein the setsof editing cassettes comprise one or more editing gRNA and donor DNApairs wherein each editing gRNA and donor DNA pair is under the controlof a first inducible promoter; assembling the editing cassettes into avector backbone thereby forming editing vectors, wherein the vectorbackbone comprises a first selectable marker, and a curing targetsequence; making cells of choice electrocompetent, wherein the cells ofchoice comprise an engine vector and the engine vector comprises acuring gRNA under the control of a second inducible promoter, a nucleaseunder the control of the third inducible promoter; and a secondselectable marker; transforming the cells of choice with a first setediting vectors to produce transformed cells; selecting for transformedcells via the first and second selectable markers; inducing editing inthe selected cells by inducing the first and third inducible promotersthereby inducing transcription of the one or more editing gRNA and donorDNA pairs and the nuclease; growing the cells until the cells reach astationary phase of growth; curing the editing vector by inducing thethird and second inducible promoters thereby inducing transcription ofthe nuclease and curing gRNA; growing the cells; rendering the cellselectrocompetent; and transforming the cells with a second set of theediting vectors to produce second transformed cells, wherein the secondset of editing vectors comprises editing cassettes with one or more gRNAand donor DNA pairs under the control of the first inducible promoter, athird selectable marker, and the curing target sequence.

In some aspects of this embodiment, the first inducible promoter and thethird inducible promoters are the same inducible promoter, and in someaspects, the first and third inducible promoters are pL promoters andeither the editing vector or the engine vector comprises a c1857 geneunder the control of a constitutive promoter.

In some aspects, the target curing sequence is a pUC origin ofreplication, and the curing gRNA is an anti-pUC origin gRNA.

In some aspects, the second inducible promoter is a pPhIF promoter.

In some aspects, the method further comprises, after the secondtransforming step, the additional steps of: selecting for the secondtransformed cells via the second and third selectable markers; inducingediting in the selected cells by inducing the first and third induciblepromoter thereby inducing transcription of the one or more editing gRNAand donor DNA pairs and the nuclease; growing the induced cells untilthe cells reach a stationary phase of growth; curing the editing vectorsfrom the second set of editing vectors in the induced cells by inducingthe third and second inducible promoters thereby inducing transcriptionof the nuclease and curing gRNA; growing the cells; rendering the cellselectrocompetent; and transforming the cells with a third set of theediting vectors to produce third transformed cells, wherein the thirdset of editing vectors comprises editing cassettes with one or more gRNAand donor DNA pairs under the control of the first inducible promoter, afourth selectable marker, and the curing target sequence.

In other aspects, the method further comprises after the thirdtransforming step, the steps of: selecting for the third transformedcells via the second and fourth selectable markers; inducing editing inthe selected cells by inducing the first and third inducible promoterthereby inducing transcription of the one or more editing gRNA and donorDNA pairs and the nuclease; growing the induced cells until the cellsreach a stationary phase of growth; curing the editing vectors from thethird set of editing vectors in the induced cells by inducing the thirdand second inducible promoters thereby inducing transcription of thenuclease and curing gRNA; growing the cells; rendering the cellselectrocompetent; and transforming the cells with a fourth set of theediting vectors to produce fourth transformed cells, wherein the fourthset of editing vectors comprises editing cassettes with one or moregRNAs and donor DNA pairs under the control of the first induciblepromoter, a fifth selectable marker, and the curing target sequence.

In some aspects, the first, second, third and fourth sets of editingcassettes each comprise a library of editing gRNA and donor DNA pairs;and in some aspects, the libraries of editing vectors each comprises atleast 1000 different editing gRNA and donor DNA pairs.

Other embodiments provide a method for curing cells during recursivenucleic acid-directed nuclease editing comprising: designing andsynthesizing sets of editing cassettes, wherein the sets of editingcassettes comprise one or more editing gRNA and donor DNA pairs whereineach editing gRNA and donor DNA pair is under the control of a firstinducible promoter; assembling the editing cassettes into a vectorbackbone thereby forming editing vectors, wherein the vector backbonecomprises a first selectable marker, a curing target sequence, and acuring gRNA under the control of a second inducible promoter; makingcells of choice electrocompetent, wherein the cells of choice comprisean engine vector and the engine vector comprises a nuclease under thecontrol of the third inducible promoter, and a second selectable marker;transforming the cells of choice with a first set editing vectors toproduce transformed cells; selecting for transformed cells via the firstand second selectable markers; inducing editing in the selected cells byinducing the first and third inducible promoters thereby inducingtranscription of the one or more editing gRNA and donor DNA pairs andnuclease; growing the cells until the cells reach a stationary phase ofgrowth; curing the editing vector by inducing the third and secondinducible promoters thereby inducing transcription of the nuclease andcuring gRNA; growing the cells; rendering the cells electrocompetent;and transforming the cells with a second set of the editing vectors toproduce second transformed cells, wherein the second set of editingvectors comprises editing cassettes with one or more gRNA and donor DNApairs under the control of the first inducible promoter, a thirdselectable marker, the curing target sequence, and the curing gRNA underthe control of the second inducible promoter.

In some aspects of this embodiment, the first inducible promoter and thethird inducible promoters are the same inducible promoter, and in someaspects, the first and third inducible promoters are pL promoters andeither the editing vector or the engine vector comprises a c1857 geneunder the control of a constitutive promoter.

In some aspects, the target curing sequence is a pUC origin ofreplication, and the curing gRNA is an anti-pUC origin gRNA.

In some aspects, the second inducible promoter is a pPhIF promoter.

In some aspects of this embodiment, the method further comprises, afterthe second transforming step, the additional steps of: selecting for thesecond transformed cells via the second and third selectable markers;inducing editing in the selected cells by inducing the first and thirdinducible promoter thereby inducing transcription of the one or moreediting gRNA and donor DNA pairs and the nuclease; growing the inducedcells until the cells reach a stationary phase of growth; curing theediting vectors from the second set of editing vectors in the inducedcells by inducing the third and second inducible promoters therebyinducing transcription of the nuclease and curing gRNA; growing thecells; rendering the cells electrocompetent; and transforming the cellswith a third set of the editing vectors to produce third transformedcells, wherein the third set of editing vectors comprises editingcassettes with one or more gRNA and donor DNA pairs under the control ofthe first inducible promoter, a fourth selectable marker, the curingtarget sequence, and the curing gRNA under the control of the secondinducible promoter.

In yet another aspect, the method may further comprise the steps of,after the third transforming step: selecting for the third transformedcells via the second and fourth selectable markers; inducing editing inthe selected cells by inducing the first and third inducible promoterthereby inducing transcription of the one or more editing gRNA and donorDNA pairs and the nuclease; growing the induced cells until the cellsreach a stationary phase of growth; curing the editing vectors from thethird set of editing vectors in the induced cells by inducing the thirdand second inducible promoters thereby inducing transcription of thenuclease and curing gRNA; growing the cells; rendering the cellselectrocompetent; and transforming the cells with a fourth set of theediting vectors to produce fourth transformed cells, wherein the fourthset of editing vectors comprises editing cassettes with one or more gRNAand donor DNA pairs under the control of the first inducible promoter, afifth selectable marker, the curing target sequence, and the curing gRNAunder the control of the second inducible promoter.

In some aspects of any of the methods, the method further comprisesbetween the transforming step and inducing step, singulating the cellsin a SWIIN, and wherein the selecting, inducing, growing, and curingsteps are performed in the SWIIN.

In some aspects, the first, second, third and fourth sets of editingcassettes each comprise a library of editing gRNA and donor DNA pairs;and in some aspects, the libraries of editing vectors each comprises atleast 1000 different editing gRNA and donor DNA pairs.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is a flow chart showing steps for an exemplary curing methodaccording to the present disclosure. FIG. 1B is an exemplary growthcurve for cells. FIG. 1C depicts an exemplary plasmid architecture forengine vector curing of an editing vector and FIG. 1D depicts anexemplary plasmid architecture for self-curing of an editing vector.FIG. 1E depicts an exemplary recursive method using a standard platingprotocol. FIG. 1F depicts the exemplary recursive method using thestandard plating protocol of FIG. 1E. FIG. 1G depicts an exemplaryrecursive method using a bulk liquid editing protocol. FIG. 1H depictsthe exemplary recursive method using the bulk liquid editing protocol ofFIG. 1G. FIG. 1I depicts an exemplary recursive method using a solidwall isolation device.

FIGS. 2A-2C depict three different views of an exemplary automatedmulti-module cell processing instrument for performing nucleicacid-guided nuclease editing.

FIG. 3A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein and in relation to FIGS. 3B-3D.FIG. 3B illustrates a perspective view of one embodiment of a rotatinggrowth vial in a cell growth module housing. FIG. 3C depicts a cut-awayview of the cell growth module from FIG. 3B. FIG. 3D illustrates thecell growth module of FIG. 3B coupled to LED, detector, and temperatureregulating components.

FIG. 4A depicts retentate (top) and permeate (middle) members for use ina tangential flow filtration module (e.g., cell growth and/orconcentration module), as well as the retentate and permeate membersassembled into a tangential flow assembly (bottom). FIG. 4B depicts twoside perspective views of a reservoir assembly of a tangential flowfiltration module. FIGS. 4C-4E depict an exemplary top, with fluidic andpneumatic ports and gasket suitable for the reservoir assemblies shownin FIG. 4B.

FIGS. 5A and 5B depict the structure and components of an embodiment ofa reagent cartridge. FIG. 5C is a top perspective view of one embodimentof an exemplary flow-through electroporation device that may be part ofa reagent cartridge. FIG. 5D depicts a bottom perspective view of oneembodiment of an exemplary flow-through electroporation device that maybe part of a reagent cartridge. FIGS. 5E-5G depict a top perspectiveview, a top view of a cross section, and a side perspective view of across section of an FTEP device useful in a multi-module automated cellprocessing instrument such as that shown in FIGS. 2A-2C.

FIG. 6A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells in a solid wall device. FIGS. 6B-6D depictan embodiment of a solid wall isolation incubation and normalization(SWIIN) module. FIG. 6E depicts the embodiment of the SWIIN module inFIGS. 6B-6D further comprising a heater and a heated cover.

FIG. 7 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module for recursive cellediting.

FIG. 8 is a simplified process diagram of an alternative embodiment ofan exemplary automated multi-module cell processing instrument usefulfor recursive cell editing.

FIG. 9 is a graph demonstrating the effectiveness of a 2-paddle rotatinggrowth vial and cell growth device as described herein for growing anEC23 cell culture vs. a conventional cell shaker.

FIG. 10 is a graph demonstrating the effectiveness of a 3-paddlerotating growth vial and cell growth device as described herein forgrowing an EC23 cell culture vs. a conventional cell shaker.

FIG. 11 is a graph demonstrating the effectiveness of a 4-paddlerotating growth vial and cell growth device as described herein forgrowing an EC138 cell culture vs. a conventional orbital cell shaker.

FIG. 12 is a graph demonstrating the effectiveness of a 2-paddlerotating growth vial and cell growth device as described herein forgrowing an EC138 cell culture vs. a conventional orbital cell shaker.

FIG. 13 is a graph demonstrating real-time monitoring of growth of anEC138 cell culture to OD₆₀₀ employing the cell growth device asdescribed herein where a 2-paddle rotating growth vial was used.

FIG. 14 is a graph demonstrating real-time monitoring of growth of s288cyeast cell culture OD₆₀₀ employing the cell growth device as describedherein where a 2-paddle rotating growth vial was used.

FIG. 15A is a graph plotting filtrate conductivity against filterprocessing time for an E. coli culture processed in the cellconcentration device/module described herein. FIG. 15B is a graphplotting filtrate conductivity against filter processing time for ayeast culture processed in the cell concentration device/moduledescribed herein.

FIG. 16A is a bar graph showing the results of electroporation of E.coli using a device of the disclosure and a comparator electroporationdevice. FIG. 16B is a bar graph showing uptake, cutting, and editingefficiencies of E. coli cells transformed via an FTEP as describedherein benchmarked against a comparator electroporation device.

FIG. 17 is a bar graph showing the results of electroporation of S.cerevisiae using an FTEP device of the disclosure and a comparatorelectroporation method.

FIG. 18 is a graph showing the editing results obtained via the liquidbulk method for increasing observed editing in live cells.

FIG. 19 is a graph comparing the percentage of editing obtained for astandard plating protocol (SPP), and replicate samples using twodifferent conditions in a solid wall isolation, induction, andnormalization device (SWIIN): the first with LB+arabinose; and thesecond with SOB followed by SOB+arabinose.

FIGS. 20A, 20B, and 20C are graphs of results obtained in experimentstesting editing efficiency and curing efficiency in engine vector-drivenrecursive experiments performed by a standard plating protocol, a bulkliquid protocol, and a SWIIN protocol.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, California (1992),all of which are herein incorporated in their entirety by reference forall purposes. Nucleic acid-guided nuclease techniques can be found in,e.g., Genome Editing and Engineering from TALENs and CRISPRs toMolecular Surgery, Appasani and Church (2018); and CRISPR: Methods andProtocols, Lindgren and Charpentier (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus (e.g., a target genomicDNA sequence or cellular target sequence) by homologous recombinationusing nucleic acid-guided nucleases. For homology-directed repair, thedonor DNA must have sufficient homology to the regions flanking the “cutsite” or site to be edited in the genomic target sequence. The length ofthe homology arm(s) will depend on, e.g., the type and size of themodification being made. The donor DNA will have two regions of sequencehomology (e.g., two homology arms) to the genomic target locus.Preferably, an “insert” region or “DNA sequence modification” region—thenucleic acid modification that one desires to be introduced into agenome target locus in a cell—will be located between two regions ofhomology. The DNA sequence modification may change one or more bases ofthe target genomic DNA sequence at one specific site or multiplespecific sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more basepairs of the genomic target sequence. A deletion or insertion may be adeletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the genomic targetsequence.

As used herein, “enrichment” refers to enriching for edited cells bysingulation, inducing editing, and growth of singulated cells intoterminal-sized colonies (e.g., saturation or normalization of colonygrowth).

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease. The term“editing gRNA” refers to the gRNA used to edit a target sequence in acell, typically a sequence endogenous to the cell. The term “curinggRNA” refers to the gRNA used to target the curing target sequence onthe editing vector.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible, and in someembodiments—particularly many embodiments such as those describedherein—the transcription of at least one component of the nucleicacid-guided nuclease editing system—and typically at least threecomponents of the nucleic acid-guided nuclease editing system—is underthe control of an inducible promoter.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, nourseothricin N-acetyl transferase, chloramphenicol,erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,rifampicin, puromycin, hygromycin, blasticidin, and G418 may beemployed. In other embodiments, selectable markers include, but are notlimited to sugars such as rhamnose, human nerve growth factor receptor(detected with a MAb, such as described in U.S. Pat. No. 6,365,373);truncated human growth factor receptor (detected with MAb); mutant humandihydrofolate reductase (DHFR; fluorescent MTX substrate available);secreted alkaline phosphatase (SEAP; fluorescent substrate available);human thymidylate synthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+cells); CD24 cell surfaceantigen in hematopoietic stem cells; human CAD gene to confer resistanceto N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2α; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers.

The term “specifically binds” as used herein includes an interactionbetween two molecules, e.g., an engineered peptide antigen and a bindingtarget, with a binding affinity represented by a dissociation constantof about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about10⁻¹¹M, about 10⁻¹²M, about 10⁻¹³M, about 10⁻¹⁴M or about 10⁻¹⁵M

The terms “target genomic DNA sequence”, “cellular target sequence”, or“genomic target locus” refer to any locus in vitro or in vivo, or in anucleic acid (e.g., genome) of a cell or population of cells, in which achange of at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The cellular target sequence can be a genomiclocus or extrachromosomal locus. The term “curing target sequence”refers to a sequence in the editing vector that is cleaved or cut tocure or clear the editing vector. The term “target sequence” refers toeither or both of a cellular target sequence and a curing targetsequence.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. As usedherein, the phrase “engine vector” comprises a coding sequence for anuclease to be used in the nucleic acid-guided nuclease systems andmethods of the present disclosure. The engine vector may also comprise,in a bacterial system, the λ Red recombineering system or an equivalentthereof. Engine vectors also typically comprise a selectable marker. Asused herein the phrase “editing vector” comprises a donor nucleic acid,including an alteration to the cellular target sequence that preventsnuclease binding at a PAM or spacer in the cellular target sequenceafter editing has taken place, and a coding sequence for a gRNA. Theediting vector may also and preferably does comprise a selectable markerand/or a barcode. In some embodiments, the engine vector and editingvector may be combined; that is, all editing and selection componentsmay be found on a single vector. Further, the engine and editing vectorscomprise control sequences operably linked to, e.g., the nuclease codingsequence, recombineering system coding sequences (if present), donornucleic acid, guide nucleic acid(s), and selectable marker(s).

Nuclease-Directed Genome Editing Generally

In preferred embodiments, the automated instrument described hereinperforms recursive nuclease-directed genome editing methods forintroducing edits to a population of cells, where editing vectors fromprevious rounds of editing are cured (e.g., cleared) before a subsequentediting vector is introduced into the population of cells. A recentdiscovery for editing live cells involves nucleic acid-guided nuclease(e.g., RNA-guided nuclease) editing. A nucleic acid-guided nucleasecomplexed with an appropriate synthetic guide nucleic acid in a cell cancut the genome of the cell at a desired location. The guide nucleic acidhelps the nucleic acid-guided nuclease recognize and cut the DNA at aspecific target sequence (either a cellular target sequence or a curingtarget sequence). By manipulating the nucleotide sequence of the guidenucleic acid, the nucleic acid-guided nuclease may be programmed totarget any DNA sequence for cleavage as long as an appropriateprotospacer adjacent motif (PAM) is nearby. In certain aspects, thenucleic acid-guided nuclease editing system may use two separate guidenucleic acid molecules that combine to function as a guide nucleic acid,e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).In other aspects, the guide nucleic acid may be a single guide nucleicacid that includes both the crRNA and tracrRNA sequences.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA may beencoded by a DNA sequence on a polynucleotide molecule such as aplasmid, linear construct, or the coding sequence may and preferablydoes reside within an editing cassette and is under the control of aninducible promoter as described below. For additional informationregarding “CREATE” editing cassettes, see U.S. Pat. Nos. 9,982,278;10,266,849; 10,240,167; 10,351,877; 10,364,442; 10,435,715; and10,465,207 and U.S. Ser. Nos. 16/551,517; 16,773,618; and 16,773,712,all of which are incorporated by reference herein.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the present methods and compositions, the guide nucleic acids areprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript under the control of an inducible promoter. The guide nucleicacids are engineered to target a desired target sequence (eithercellular target sequence or curing target sequence) by altering theguide sequence so that the guide sequence is complementary to a desiredtarget sequence, thereby allowing hybridization between the guidesequence and the target sequence. In general, to generate an edit in thetarget sequence, the gRNA/nuclease complex binds to a target sequence asdetermined by the guide RNA, and the nuclease recognizes a protospaceradjacent motif (PAM) sequence adjacent to the target sequence. Thetarget sequence can be any polynucleotide endogenous or exogenous to aprokaryotic or eukaryotic cell, or in vitro. For example, the targetsequence can be a polynucleotide residing in the nucleus of a eukaryoticcell. A target sequence can be a sequence encoding a gene product (e.g.,a protein) or a non-coding sequence (e.g., a regulatory polynucleotide,an intron, a PAM, or “junk” DNA) or a curing target sequence in anediting vector. In the present description, the target sequence for oneof the gRNAs, the curing gRNA, is on the editing vector.

The editing guide nucleic acid may be and preferably is part of anediting cassette that encodes the donor nucleic acid that targets acellular target sequence. Alternatively, the editing guide nucleic acidmay not be part of the editing cassette and instead may be encoded onthe editing vector backbone. For example, a sequence coding for anediting guide nucleic acid can be assembled or inserted into a vectorbackbone first, followed by insertion of the donor nucleic acid in,e.g., an editing cassette. In other cases, the donor nucleic acid in,e.g., an editing cassette can be inserted or assembled into a vectorbackbone first, followed by insertion of the sequence coding for theediting guide nucleic acid. Preferably, the sequence encoding theediting guide nucleic acid and the donor nucleic acid are locatedtogether in a rationally-designed editing cassette and aresimultaneously inserted or assembled into a vector backbone to create anediting vector. In yet other embodiments, the sequence encoding theguide nucleic acid and the sequence encoding the donor nucleic acid areboth included in the editing cassette.

The target sequence—both the cellular target sequence and the curingtarget sequence—is associated with a proto-spacer mutation (PAM), whichis a short nucleotide sequence recognized by the gRNA/nuclease complex.The precise preferred PAM sequence and length requirements for differentnucleic acid-guided nucleases vary; however, PAMs typically are 2-7base-pair sequences adjacent or in proximity to the target sequence and,depending on the nuclease, can be 5′ or 3′ to the target sequence.Engineering of the PAM-interacting domain of a nucleic acid-guidednuclease may allow for alteration of PAM specificity, improve targetsite recognition fidelity, decrease target site recognition fidelity, orincrease the versatility of a nucleic acid-guided nuclease.

In certain embodiments, the genome editing of a cellular target sequenceboth introduces a desired DNA change to a cellular target sequence,e.g., the genomic DNA of a cell, and removes, mutates, or rendersinactive a proto-spacer mutation (PAM) region in the cellular targetsequence. Rendering the PAM at the cellular target sequence inactiveprecludes additional editing of the cell genome at that cellular targetsequence, e.g., upon subsequent exposure to a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid in later roundsof editing. Thus, cells having the desired cellular target sequence editand an altered PAM can be selected for by using a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid complementary tothe cellular target sequence. Cells that did not undergo the firstediting event will be cut rendering a double-stranded DNA break, andthus will not continue to be viable. The cells containing the desiredcellular target sequence edit and PAM alteration will not be cut, asthese edited cells no longer contain the necessary PAM site and willcontinue to grow and propagate.

The range of target sequences (both cellular target sequences and curingtarget sequences) that nucleic acid-guided nucleases can recognize isconstrained by the need for a specific PAM to be located near thedesired target sequence. As a result, it often can be difficult totarget edits with the precision that is necessary for genome editing. Ithas been found that nucleases can recognize some PAMs very well (e.g.,canonical PAMs), and other PAMs less well or poorly (e.g., non-canonicalPAMs). Because the methods disclosed herein allow for identification ofedited cells in a background of unedited cells, the methods allow foridentification of edited cells where the PAM is less than optimal; thatis, the methods for identifying edited cells herein allow foridentification of edited cells even if editing efficiency is very low.Additionally, the present methods expand the scope of target sequencesthat may be edited since edits are more readily identified, includingcells where the genome edits are associated with less functional PAMs.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcell types, such as archaeal, prokaryotic or eukaryotic cells.Eukaryotic cells can be yeast, fungi, algae, plant, animal, or humancells. Eukaryotic cells may be those of or derived from a particularorganism, such as a mammal, including but not limited to human, mouse,rat, rabbit, dog, or non-human mammals including non-human primates. Thechoice of nucleic acid-guided nuclease to be employed depends on manyfactors, such as what type of edit is to be made in the target sequenceand whether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymesand nuclease fusions thereof. Nuclease fusion enzymes typically comprisea CRISPR nucleic acid-guided nuclease engineered to cut one DNA strandin the target DNA rather than making a double-stranded cut, and thenuclease portion is fused to a reverse transcriptase. For moreinformation on nickases and nuclease fusion editing see U.S. Ser. Nos.16/740,418; 16/740,420 and 16/740,421, all filed 11 Jan. 2020. As withthe guide nucleic acid, the nuclease is encoded by a DNA sequence on avector (e.g., the engine vector) and be under the control of aninducible promoter. In some embodiments, the inducible promoter may beseparate from but the same as the inducible promoter controllingtranscription of the guide nucleic acid; that is, a separate induciblepromoter drives the transcription of the nuclease or nuclease fusionandguide nucleic acid sequences but the two inducible promoters may be thesame type of inducible promoter (e.g., both are pL promoters).Alternatively, the inducible promoter controlling expression of thenuclease may be different from the inducible promoter controllingtranscription of the guide nucleic acid; that is, e.g., the nuclease maybe under the control of the pBAD inducible promoter, and the guidenucleic acid may be under the control of the pL inducible promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid comprising homology to the cellular target sequence.In some embodiments, the donor nucleic acid is on the samepolynucleotide (e.g., editing vector or editing cassette) as the guidenucleic acid and preferably is (but not necessarily is) under thecontrol of the same promoter as the editing gRNA (e.g., a singlepromoter driving the transcription of both the editing gRNA and thedonor nucleic acid). The donor nucleic acid is designed to serve as atemplate for homologous recombination with a cellular target sequencenicked or cleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the cellular target sequence (e.g., a homology arm). Whenoptimally aligned, the donor nucleic acid overlaps with (iscomplementary to) the cellular target sequence by, e.g., about 20, 25,30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. The donor nucleicacid comprises two homology arms (regions complementary to the cellulartarget sequence) flanking the mutation or difference between the donornucleic acid and the cellular target sequencee. The donor nucleic acidcomprises at least one mutation or alteration compared to the cellulartarget sequence, such as an insertion, deletion, modification, or anycombination thereof compared to the cellular target sequence.

Again, the donor nucleic acid is preferably provided as part of arationally-designed editing cassette, which is inserted into an editingvector backbone where the editing vector backbone may comprise apromoter driving transcription of the editing gRNA and the donor DNA,and also comprise a selectable marker different from the selectablemarker contained on the engine vector, as well as a curing targetsequence that is cut or cleaved during curing. Moreover, there may bemore than one, e.g., two, three, four, or more editing gRNA/donornucleic acid rationally-designed editing cassettes inserted into anediting vector (alternatively, a single rationally-designed editingcassette may comprise two to several editing gRNA/donor DNA pairs),where each editing gRNA is under the control of separate differentpromoters, separate like promoters, or where all gRNAs/donor nucleicacid pairs are under the control of a single promoter. In preferredembodiments the promoter driving transcription of the editing gRNA andthe donor nucleic acid (or driving more than one editing gRNA/donornucleic acid pair) is an inducible promoter and the promoter drivingtranscription of the nuclease or nuclease fusion is an induciblepromoter as well. In some embodiments and preferably, the nuclease andediting gRNA/donor DNA are under the control of the same induciblepromoter.

Inducible editing is advantageous in that singulated cells can be grownfor several to many cell doublings before editing is initiated, whichincreases the likelihood that cells with edits will survive, as thedouble-strand cuts caused by active editing are largely toxic to thecells. This toxicity results both in cell death in the edited colonies,as well as possibly a lag in growth for the edited cells that do survivebut must repair and recover following editing. However, once the editedcells have a chance to recover, the size of the colonies of the editedcells will eventually catch up to the size of the colonies of uneditedcells. It is this toxicity, however, that is exploited herein to performcuring.

In addition to the donor nucleic acid, an editing cassette may compriseand preferably does comprise one or more primer sites. The primer sitescan be used to amplify the editing cassette by using oligonucleotideprimers; for example, if the primer sites flank one or more of the othercomponents of the editing cassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a cellular targetsequence—one or more PAM sequence alterations that mutate, delete orrender inactive the PAM site in the cellular target sequence. The PAMsequence alteration in the cellular target sequence renders the PAM site“immune” to the nucleic acid-guided nuclease and protects the cellulartarget sequence from further editing in subsequent rounds of editing ifthe same nuclease is used.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding cellulartarget sequence. The barcode typically comprises four or morenucleotides. In some embodiments, the editing cassettes comprise acollection or library editing gRNAs and of donor nucleic acidsrepresenting, e.g., gene-wide or genome-wide libraries of editing gRNAsand donor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a nucleic acid-guided nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineerednuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination.

The engine and editing vectors comprise control sequences operablylinked to the component sequences to be transcribed. As stated above,the promoters driving transcription of one or more components of thenucleic acid-guided nuclease editing system preferably are inducible. Anumber of gene regulation control systems have been developed for thecontrolled expression of genes in plant, microbe, and animal cells,including mammalian cells, including the pL promoter (induced by heatinactivation of the cI857 repressor), the pPhIF promoter (induced by theaddition of 2,4 diacetylphloroglucinol (DAPG)), the pBAD promoter(induced by the addition of arabinose to the cell growth medium), andthe rhamnose inducible promoter (induced by the addition of rhamnose tothe cell growth medium). Other systems include thetetracycline-controlled transcriptional activation system(Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen,PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur etal., Strategies 5(3):70-72 (1992); U.S. Pat. No. 4,833,080), theecdysone-inducible gene expression system (No et al., PNAS,93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al.,BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible geneexpression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) aswell as others. In the present methods used in the modules andinstruments described herein, it is preferred that at least one of thenucleic acid-guided nuclease editing components (e.g., the nucleaseand/or the gRNA) is under the control of a promoter that is activated bya rise in temperature, as such a promoter allows for the promoter to beactivated by an increase in temperature, and de-activated by a decreasein temperature, thereby “turning off” the editing process. Thus, in thescenario of a promoter that is de-activated by a decrease intemperature, editing in the cell can be turned off without having tochange media; to remove, e.g., an inducible biochemical in the mediumthat is used to induce editing.

Curing

“Curing” is a process in which a vector—here, the editing vector used ina prior round of editing or an engine vector after the final round ofediting—is eliminated from the cells being edited. Curing can beaccomplished by 1) cleaving the editing vector using a curing gRNA onthe engine or editing vectors thereby rendering the editing vectornonfunctional; 2) diluting the editing vector in the cell population viacell growth—that is, the more growth cycles the cells go through inmedium without the antibiotic that selects for the editing vector thefewer daughter cells will retain the editing or engine vector(s)); or 3)by utilizing a heat-sensitive origin of replication on the editingvector. The present disclosure is drawn to transcribing a curing gRNAlocated on either the editing vector (“self cure”) or the engine vector(“engine cure”) to cut or cleave a locus located in a curing targetsequence in the editing vector after a round of editing and before anext round of editing in a recursive editing process.

FIG. 1A is a flow chart for the curing methods 100 according to thepresent disclosure. In a first step, a library of rationally-designedediting cassettes is synthesized 102. Methods and compositionsparticularly favored for designing and synthesizing editing cassettesare described in U.S. Pat. Nos. 9,982,278; 10,266,849; 10,240,167;10,351,877; 10,364,442; 10,435,715; and 10,465,207 and U.S. Ser. Nos.16/551,517; 16,773,618; and 16,773,712, all of which are incorporated byreference herein.

Once designed and synthesized, the editing cassettes are amplified,purified and assembled into a vector backbone 104 to create editingcassettes. A number of methods may be used to assemble the editingcassettes including Gibson Assembly®, CPEC, SLIC, Ligase Cycling etc.Additional assembly methods include gap repair in yeast (Bessa, Yeast,29(10):419-23 (2012)), gateway cloning (Ohtsuka, Curr Pharm Biotechnol,10(2):244-51 (2009); US Patent No. 5,888,732 to Hartley et al.; U.S.Pat. No. 6,277,608 to Hartley et al.; and topoisomerase-mediated cloning(Udo, PLoS One, 10(9):e0139349 (2015)); U.S. Pat. No. 6,916,632 B2 toChestnut et al. These and other nucleic acid assembly techniques aredescribed, e.g., in Sands and Brent, Curr Protoc Mol Biol.,113:3.26.1-3.26.20 (2016); Casini et al., Nat Rev Mol Cell Biol.,(9):568-76 (2015); and Patron, Curr Opinion Plant Biol., 19:14-9(2014)).

In addition to preparing editing cassettes, cells of choice are madeelectrocompetent 120 for transformation. The cells that can be editedinclude any prokaryotic, archaeal or eukaryotic cell. For example,prokaryotic cells for use with the present illustrative embodiments canbe gram positive bacterial cells, e.g., Bacillus subtilis, orgram-negative bacterial cells, e.g., E. coli cells. Eukaryotic cells foruse with the automated multi-module cell editing instruments of theillustrative embodiments include any plant cells and any animal cells,e.g. fungal cells, insect cells, amphibian cells nematode cells, ormammalian cells.

Once the cells of choice are rendered electrocompetent 120, the cellsand editing vectors are combined and the editing vectors are transformedinto (e.g., electroporated into) the cells 106. The cells may be alsotransformed simultaneously with a separate engine vector expressing anediting nuclease; alternatively and preferably, the cells may alreadyhave been transformed with an engine vector configured to express thenuclease; that is, the cells may have already been transformed with anengine vector or the coding sequence for the nuclease may be stablyintegrated into the cellular genome such that only the editing vectorneeds to be transformed into the cells.

Transformation is intended to include to a variety of art-recognizedtechniques for introducing an exogenous nucleic acid sequence (e.g.,DNA) into a target cell, and the term “transformation” as used hereinincludes all transformation and transfection techniques. Such methodsinclude, but are not limited to, electroporation, lipofection,optoporation, injection, microprecipitation, microinjection, liposomes,particle bombardment, sonoporation, laser-induced poration, headtransfection, calcium phosphate or calcium chloride co-precipitation, orDEAE-dextran-mediated transfection. Cells can also be prepared forvector uptake using, e.g., a sucrose or glycerol wash. Additionally,hybrid techniques that exploit the capabilities of mechanical andchemical transfection methods can he used, e.g., magnetofection, atransfection methodology that combines chemical transfection withmechanical methods. In another example, cationic lipids may he deployedin combination with gene guns or electroporators. Suitable materials andmethods for transforming or transfecting target cells can be found,e.g., in Green and Sambrook, Molecular Cloning: A Laboratory Manual,4th, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2014). The present automated methods using the automated multi-modulecell processing instrument utilize flow-through electroporation such asthe exemplary device shown in FIGS. 5C-5G.

Once transformed, the cells are allowed to recover and selection isperformed 108 to select for cells transformed with the editing vector,which in addition to an editing cassette comprises an appropriateselectable marker. As described above, drug selectable markers such asampilcillin/carbenicillin, kanamycin, chloramphenicol, nourseothricinN-acetyl transferase, erythromycin, tetracycline, gentamicin, bleomycin,streptomycin, puromycin, hygromycin, blasticidin, and G418 or otherselectable markers may be employed.

Following selection for properly transformed cells, editing is induced110 in the cells by induction of transcription of one or both—preferablyboth—of the nuclease and gRNA. Induction of transcription of one, or,preferably both, of the nuclease and gRNA is prompted by, e.g., using apL promoter system where the pL promoter is induced by raising thetemperature of the cells in the medium to 42° C. for, e.g., one to manyhours to induce expression of the nuclease and gRNA for cutting andediting. A number of gene regulation control systems have been developedfor the controlled expression of genes in plant, microbe, and animalcells, including mammalian cells, including, in addition to the pLpromoter, the pPhIF promoter (induced by the addition of 2,4diacetylphloroglucinol (DAPG)), the pBAD promoter (induced by theaddition of arabinose to the cell growth medium), and the rhamnoseinducible promoter (induced by the addition of rhamnose to the cellgrowth medium). Other systems include the tetracycline-controlledtranscriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (PaloAlto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), theLac Switch Inducible system (Wyborski et al., Environ Mol Mutagen,28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S.Pat. No. 4,833,080), the ecdysone-inducible gene expression system (Noet al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system(Mullick et al., BMC Biotechnology, 6:43 (2006)), and thetamoxifen-inducible gene expression (Zhang et al., Nucleic AcidsResearch, 24:543-548 (1996)) as well as others.

The present compositions and methods preferably make use ofrationally-designed editing cassettes such as CREATE cassettes, asdescribed above. Each editing cassette comprises an editing gRNA, adonor DNA comprising an intended edit and a PAM or spacer mutation;thus, e.g., a two-cassette multiplex editing cassette comprises a firstediting gRNA, a first editing donor DNA, and a first intended edit and afirst PAM or spacer mutation, and at least a second editing gRNA, atleast a second donor DNA, and at least a second intended edit and asecond PAM or spacer mutation. In some embodiments, a single promotermay drive transcription of both the first and second editing gRNAs andboth the first and second donor DNAs, and in some embodiments, separatepromoters may drive transcription of the first editing gRNA and firstdonor DNA, and transcription of the second editing gRNA and second donorDNA. In addition, multiplex editing cassettes may comprise nucleic acidelements between the editing cassettes with, e.g., primer sequences,bridging oligonucleotides, and other “cassette-connecting” sequenceelements that allow for the assembly of the multiplex editing cassettes.

Once editing is induced 110, the cells are grown until the cells enter(or are close to entering) the stationary phase of growth 112, followedby inducing curing of the editing vector 114 by activating an induciblepromoter driving transcription of the curing gRNA and inducing theinducible promoter driving transcription of the nuclease. It has beenfound that curing is particularly effective if the edited cells are inthe stationary phase of growth. In yet some aspects, the cells are grownfor at least 75% of log phase, 80% of log phase, 85% of log phase, 90%of log phase, 95% of log phase, or are in a stationary phase of growthbefore inducing curing. Exemplary genetic and inducing components forinducing curing are described in more detail in relation to FIGS. 1C and1D. Once the editing vector has been cured 114, the cells are allowed torecover and grow, and then the cells are made electrocompetent 116 onceagain, ready for another round of editing 118.

FIG. 1B depicts a typical growth curve 150 for cells in culture (opticaldensity versus time). Initially there is a lag phase 151, then the cellsenter log phase 152 where they grow quickly, and finally the cells reachstationary phase 154 where the cells are no longer dividing. The presentmethods employ inducing curing at timepoint 153 or later when the cellsare in the stationary phase of growth or nearly so; that is, the cellsare induced at a timepoint at least 60% into the log phase of growth, orat least 65% into the log phase of growth, or at least 70% into the logphase of growth, or at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 79, 98, or99% into the log phase of growth, and at any time during the stationaryphase of growth.

FIG. 1C depicts an exemplary plasmid architecture for engine curing ofan editing vector comprising an engine vector (on left) and an editingvector (on right); that is, the sequence for the curing gRNA thattargets the curing target sequence on the editing vector is located onthe engine vector. The engine vector comprises a pBAD inducible promoter5′ of and driving a lambda Red recombineering system. The λ Redrecombineering system works as a “band aid” or repair system fordouble-strand breaks in bacteria, and in some species of bacteria the λRed recombineering system (or some other recombineering system) must bepresent for the double-strand breaks that occur during editing toresolve. In yeast and other eukaryotic cells, however, recombineeringsystems are not required. The inducible promoter (in this case pBAD, butother inducible promoters may be used) driving transcription of the λRed recombineering system components is most preferably a differentinducible promoter than that driving transcription of the nuclease andthe editing gRNA, as it is preferred that the recombineering system beactive before the nuclease is induced. That is, it is preferred that the“band aid” double-strand break repair machinery be active before thenuclease starts cutting the cellular genome.

In addition to the λ Red recombineering system, the engine vector alsocomprises an origin of replication (here an SC101 origin, which may betemperature sensitive, but need not be) 3′ of the λ Red recombineeringsystem, followed by a pPhIF inducible primer 3′ of the SC101 origindriving transcription of the curing gRNA, in this case the curing gRNAis a gRNA that renders inactive the pUC origin of replication on theediting vector (e.g., the curing target sequence). Next, 3′ of thecuring gRNA sequence is a promoter (typically a constitutive promoter)driving transcription of the c1857 repressor gene, which activelyrepresses the pL promoter at 30° C. and degrades at 42° C. therebyactivating the pL promoter. Three prime of the c1857 coding sequence isa promoter (also typically a constitutive promoter) drivingtranscription of an antibiotic resistance gene—here, a carbenicillinresistance gene—followed by a pL inducible promoter drivingtranscription of MAD7, the nuclease.

The editing vector on the right in FIG. 1C comprises a pL promoterdriving transcription of an editing cassette, where the editing cassetteincludes a coding sequence for an editing gRNA and a donor DNA sequence(e.g., a homology arm or “HA”). The donor DNA sequence—in addition to asequence for a desired edit in a nucleic acid sequence endogenous to thecell (e.g., the cellular target sequence)−often further comprises aPAM-altering sequence, which is most often a sequence that disables thePAM at the cellular target sequence in the genome. The editing vectorfurther comprises a promoter (typically a constitutive promoter) drivingtranscription of an antibiotic resistance gene (e.g., kanamycin orchloramphenicol), followed by a pUC origin of replication. The anti-pUCgRNA (e.g., the curing gRNA), whose transcription is controlled by thepPhIF inducible promoter on the engine, comprises a gRNA that disablesthe pUC origin on the editing vector (represented by an arrow andscissors in FIG. 1C).

FIG. 1D depicts an exemplary plasmid architecture for self-curing of anediting vector, comprising an engine vector (on left) and an editingvector (on right); that is, the sequence for the curing gRNA thattargets the curing target sequence on the editing vector is located onthe editing vector itself. The engine vector comprises a pBAD induciblepromoter 5′ of and driving a lambda red recombineering system. Asdescribed above, the λ Red recombineering system works as a “band aid”or repair system for double-strand breaks in bacteria. The induciblepromoter (in this case pBAD, but other inducible promoters may be used)driving transcription of the λ Red recombineering system components ismost preferably a different inducible promoter than that drivingtranscription of the nuclease and the editing gRNA, as it is preferredthat the recombineering system be active before the nuclease is induced.In addition to the λ Red recombineering system, the engine vector alsocomprises an origin of replication (here an SC101 origin, which may betemperature sensitive, but need not be) 3′ of the λ Red recombineeringsystem, followed by a promoter (typically a constitutive promoter)driving transcription of the c1857 repressor gene, which activelyrepresses the pL promoter at 30° C. and degrades at 42° C. therebyactivating the pL promoter. Three prime of the c1857 coding sequence isa promoter (also typically a constitutive promoter) drivingtranscription of a carbenicillin resistance gene, followed by a pLinducible promoter driving transcription of MAD7, the nuclease.

The editing vector on the right in FIG. 1D comprises a pL promoterdriving transcription of an editing cassette, where the editing cassetteincludes a coding sequence for an editing gRNA and a donor DNA sequence(e.g., a homology arm or “HA”). The donor DNA sequence—in addition to asequence for a desired, intended edit in a nucleic acid sequenceendogenous to the cell—further comprises a PAM-altering sequence, asequence that disables the PAM at the cellular target sequence in thegenome. The editing vector further comprises a pPhIF inducible primer 3′of the editing cassette, where the pPhIF promoter drives transcriptionof the curing gRNA. As in FIG. 1C, the curing gRNA is a gRNA thatrenders inactive the pUC origin located on the editing vector; however,in this architecture the curing gRNA is located on the editing vector,hence the editing vector is “self-curing.” Three prime of the curinggRNA sequence is a promoter, typically a constitutive promoter, drivingtranscription of an antibiotic resistance gene (e.g., kanamycin orchloramphenicol, that is an antibiotic resistance gene different fromthe antibiotic resistance gene located on the engine vector), followedby the pUC origin of replication. The anti-pUC curing gRNA, whosetranscription is controlled by the pPhIF inducible promoter, comprises acuring gRNA that cures the editing vector by disabling (e.g., cleavingor cutting) the pUC origin on the editing vector (represented by anarrow and scissors in FIG. 1D). Thus, in the system in FIG. 1D, theediting vector self cures, as the curing gRNA (e.g., the anti-pUC gRNA)is transcribed from the editing vector and disables the pUC origin ofreplication on the editing vector.

FIG. 1E depicts an exemplary recursive method 160 a using a standardplating protocol (SPP) employing, e.g., the exemplary engine and editingvectors shown in FIGS. 1C or 1D. The recursive method 160 a begins withcompetent cells 162, for example, E. coli cells that have beenpreviously transformed with an engine vector expressing a nucleicacid-guided nuclease such as MAD7 and a selective marker, such as achloramphenicol resistance gene. At step 163, the competent cells aretransformed with a library of editing vectors, where each editing vectorin the library of editing vectors comprises an editing cassette with asequence coding for an editing gRNA and a donor DNA, and each editingvector also comprises an antibiotic resistance gene such as a geneconferring resistance to carbenicillin and a curing target sequence.Following transformation, the cells are allowed to recover 164 in mediumwithout antibiotic, and then at step 165, the cells are diluted ifnecessary, or transferred into medium containing, e.g., chloramphenicoland, e.g., carbenicillin to select for both the engine and editingvectors. The cells are then plated 166 a on solid medium containing,e.g., arabinose, which induces the lambda red recombination systemencoded by, e.g., the engine vector (see, e.g., FIGS. 1C and 1D). Onceplated the cells are allowed to grow at, e.g., 30° C. for 9 hours, at42° C. for 2 hours—thereby inducing the pL promoters drivingtranscription of the nuclease on the engine vector and the editingcassette (e.g., the editing gRNA and donor DNA) on the editingvector—then at 30° C. for at least 9 more hours.

At step 167, colonies formed by the transformed cells are removed fromthe solid medium by, e.g., scraping the colonies from the medium or bypicking colonies and then the harvested cells are placed in medium,washed to remove the carbenicillin and resuspended in medium containingchloramphenicol (continuing to select for the engine vector) and allowedto grow until the cells reach the stationary phase of growth or nearlyso. Again, it has been found that curing is particularly effective ifthe edited cells are in the stationary phase of growth when curing isinduced. For example, the cells are grown until they have grown for atleast 75% of log phase, 80% of log phase, 85% of log phase, 90% of logphase, 95% of log phase, or are in a stationary phase of growth beforeinducing curing.

The editing vectors in the cells are then cured 168 by inducingtranscription of the curing gRNA that cuts the pUC origin of replicationon the editing vector. Induction of the curing gRNA is accomplished byfirst raising the temperature of the culture to 42° C. for 2 hours,thereby inducing the pL promoter driving transcription of the nuclease,and second by the addition of 2,4 diacetylphloroglucinol (DAPG) toinduce the pPhIF promoter driving transcription of the anti-pUC gRNA.After 2 hours at 42° C., the temperature of the cell culture is loweredto 30° C. thereby halting transcription of the nuclease, and the cellsare allowed to recover and grow for 6 additional hours. Theco-expression of the nuclease and the anti-pUC gRNA permits targeting ofthe pUC origin of expression on the editing vector (e.g., the curingtarget sequence). As an alternative protocol to increasing thetemperature of the culture to 42° C. combined with addition of DAPG, onecan induce the pL promoter driving transcription of the nuclease byincreasing the temperature of the culture to 42° C. for two hours, thenlower the temperature of the culture to 30° C. and add DAPG to inducetranscription of the anti-pUC gRNA; that is, the induction of thenuclease and the anti-pUC gRNA may be sequential rather thansimultaneous.

At step 169, the cells are washed with medium containing chloramphenicol(again to select for cells comprising the engine vector), and the cellsare allowed to recover. At this point, the edited cells can be grown to,e.g., OD=0.5, made electrocompetent once more 170, and be subjected to asecond round of editing 171.

FIG. 1F depicts the exemplary recursive editing and curing method usingthe standard plating protocol (SPP) of FIG. 1E. FIG. 1F depicts theexemplary standard plating protocol embodiment of an improved protocol180 for performing nucleic acid-guided nuclease genome editing andcuring using inducible promoters to drive expression of the editinggRNA, the nuclease, and the curing gRNA. In FIG. 1F as in FIG. 1E, alibrary or collection of editing vectors 182 is introduced 183 (e.g.,electroporated) into cultured cells 184 that already comprise a codingsequence for a nuclease under the control of an inducible promoter. LikeFIG. 1E (and as depicted in FIGS. 1C and 1D), the coding sequence forthe nuclease is contained on an “engine vector” with a selectable markerthat has already been transformed into the cells, although in otherembodiments, the coding sequence for the nuclease may be integrated intothe genome of the cells. In yet other embodiments, the coding sequencefor the nuclease may be located on the editing vector (that is, acombined engine and editing vector).

The editing vectors 182 comprise a donor DNA with a desired, intendededit vis-à-vis a cellular target sequence with a PAM-altering sequencewhich is most often a sequence that disables the PAM at the target sitein the genome, a coding sequence for an editing gRNA under the controlof an inducible promoter, and a selectable marker. Depending on whetherthe system is an engine curing system or a self-curing system, there isalso a coding sequence for a curing gRNA under the control of aninducible promoter located on the engine vector or editing vector,respectively.

Once the cells 184 have been transformed with the editing vectors, thecells are plated 185 on selective medium on substrate or plate 186 toselect for cells that have both the engine and the editing vectors. Thecells are diluted before plating such that the cells are substantiallyor largely singulated—separated enough so that they and the coloniesthey form are separated from other cell colonies—and the cells are thengrown 187 on plate or substrate 186 until colonies 188 begin to form.The cells are allowed to grow at, e.g., 30° C. for, e.g., between 2 and300, or between 5 and 150, or between 10 and 50 doublings, establishingclonal colonies. This initial growth of cells is to accumulate enoughclonal cells in a colony to survive induction of editing.

Once colonies are established, cutting and editing of the cellulargenome is induced by first inducing the promoter driving transcriptionof the λ Red recombineering system, and second by inducing or activatingthe promoters driving the gRNA and nuclease. The inducible promoterdriving expression of the λ Red recombineering system preferably isdifferent from the inducible promoter driving transcription of the gRNAand nuclease and preferably the λ Red recombineering system is inducedbefore induction of the nuclease and gRNA, as the λ Red recombineeringsystem works as the “band aid” or repair system for double-strand breaksin bacteria, and in some species of bacteria (but not in other celltypes such as yeast or other eukaryotic cells) must be present for thedouble-strand breaks that occur during editing to resolve. The λ Redrecombineering system may be under the control of, e.g., a pBADpromoter. The pBAD promoter is regulated (induced) by the addition ofarabinose to the growth medium. Thus, if there is arabinose contained inthe selective medium of substrate or plate 326, the λ Red recombineeringsystem will be activated when the cells are grown 327. As for inductionof editing, if transcription of the gRNA and nuclease are both undercontrol of the pL promoter, transcription of the gRNA and nuclease isinduced by increasing the temperature to 42° C. for, e.g., a half-hourto two hours (or more, depending on the cell type), which activates thepL inducible promoter. Following induction of cutting and editing and atwo-hour 42° C. incubation, the temperature is returned to 30° C. toallow the cells to recover and to disable the pL promoter system.

Once the cells have been edited and have been grown at 30° C. forseveral hours, the colonies are then pooled 189 by, e.g., scraping thecolonies off the substrate or plate to pool 190 the cells from the cellcolonies. Once the colonies are pooled, curing 191 can take place. Asdescribed in relation to FIG. 1E, the editing vectors in the cells arecured by growing the cells until they are in the stationary phase ofgrowth or nearly so, and by inducing transcription of the curing gRNAthat cuts the pUC origin of replication on the editing vector. Inductionof the anti-pUC gRNA is accomplished by first raising the temperature ofthe culture to 42° C. for 2 hours, thereby inducing the pL promoterdriving transcription of the nuclease, and second by the addition of 2,4diacetylphloroglucinol (DAPG) to induce the pPhIF promoter drivingtranscription of the anti-pUC gRNA. After 2 hours at 42° C., thetemperature of the cell culture is lowered to 30° C., and the cells areallowed to recover and grow for 6 additional hours. The co-expression ofthe nuclease and the anti-pUC gRNA permits targeting of the pUC originof expression on the editing vector thereby producing edited cells inwhich the editing vector has been cured 192. Additionally, growing thecells performs “passive” curing as well, where the editing vector is“diluted out” of the growing cell population, as removing from themedium the antibiotic that selects for the editing vector removespressure on cells to retain the editing vector. Finally, at step 193 theedited cells are allowed to recover, then are grown to, e.g., OD=0.5,and rendered electrocompetent 193 so that the cells 194 are ready foranother round of editing.

FIG. 1G depicts an exemplary recursive method 160 b using a bulk liquidediting protocol. The recursive method 160 b—as with the recursivemethod 160 a—begins with competent cells 162, for example E. coli cellsthat have been previously transformed with an engine vector expressing anucleic acid-guided nuclease such as MAD7 and a selective marker, suchas a chloramphenicol resistance gene. At step 163, the competent cellsare transformed with a library of editing vectors, where each editingvector in the library of editing vectors comprises an editing cassettewith a sequence coding for an editing gRNA and a donor DNA, and eachediting vector also comprises an antibiotic resistance gene such as agene conferring resistance to, e.g., carbenicillin, where the antibioticresistance gene located on the editing vector is different from theantibiotic resistance gene located on the engine vector. Followingtransformation, the cells are allowed to recover 164 in medium withoutantibiotic, and then at step 165, the cells are diluted, if necessary,or transferred into medium containing chloramphenicol and carbenicillinto select for both the engine and editing vectors.

After transfer to a larger liquid volume 166b for bulk liquid editing,the cells are outgrown; that is, the cells are grown to saturation (seeFIG. 1B). Like the curing step, it has been determined that editing in abulk liquid culture is optimized when the cells have been grown to latelog phase or saturation before inducing editing. Again, the cells aregrown through at least 60% of log phase, or at least 75% of log phase,80% of log phase, 85% of log phase, 90% of log phase, 95% of log phase,or are in a stationary phase of growth before inducing curing. Thus, thecells are outgrown, then arabinose is added to the medium, inducing thelambda Red recombination system encoded by, e.g., the engine vector(see, e.g., FIGS. 1C and 1D). Once the lambda Red recombination systemis induced for, e.g., 30 minutes to an hour, the temperature isincreased 42° C. for 2 hours, thereby inducing the pL promoters drivingtranscription of the nuclease on the engine vector and the editingcassette on the editing vector and thus editing. After 2 hours at 42°C., the cells are grown at 30° C. for at least 9 more hours.

At step 173, the cells are washed to, e.g., remove the carbenicillin,and resuspended in medium containing chloramphenicol (continuing toselect for the engine vector) and the cells may be grown for anotherlength of time to assure that the cells are in the stationary phase ofgrowth for curing. The editing vectors in the cells are then cured 168by inducing transcription of the editing gRNA that cuts the pUC originof replication on the editing vector. Induction of the anti-pUC gRNA isaccomplished by first raising the temperature of the culture to 42° C.for 2 hours, thereby inducing the pL promoter driving transcription ofthe nuclease, and second by the addition of 2,4 diacetylphloroglucinol(DAPG) to induce the pPhIF promoter driving transcription of theanti-pUC gRNA (see, e.g., the vector architecture of the engine andediting vectors in FIGS. 1C and 1D). After 2 hours at 42° C., thetemperature of the cell culture is lowered to 30° C., and the cells areallowed to recover and grow for 6 additional hours. The co-expression ofthe nuclease and the anti-pUC gRNA permits targeting of the pUC originof expression on the editing vector. At step 169, the cells are washedwith medium containing chloramphenicol (again to select for cellscomprising the engine vector), and the cells are allowed to recover. Atthis point, the edited cells can be grown to a proper OD (e.g., OD=0.5),made electrocompetent once more 170, and subjected to a second round ofediting 171.

FIG. 1H depicts the exemplary recursive method using the bulk liquidediting protocol of FIG. 1G. FIG. 1H depicts an exemplary protocol 1000for performing nucleic acid-guided nuclease genome editing and curing.FIG. 1H depicts the protocol 160 b shown in FIG. 1G for editing cells.First, a library or collection of editing vectors 1002 (editing vectorseach comprising an editing cassette) is introduced 1003 (e.g.,electroporated) into cultured cells 1004 that comprise a coding sequencefor a nuclease (e.g., MAD7) under the control of an inducible promoter,contained on an engine vector (along with a selectable marker) that hasalready been transformed into the cells or already integrated into thegenome of the cells being transformed. The editing vectors 1002 comprisea donor DNA comprising a PAM or spacer-altering sequence, a codingsequence for an editing gRNA under the control of an inducible promoter,and a selectable marker. Depending on whether the system is an enginecuring system or a self-curing system, a coding sequence for a curinggRNA under the control of an inducible promoter is located on the enginevector or editing vector, respectively.

At step 1005, cells are grown until they reach stationary phase, ornearly so. Once the cells reach the stationary phase 1006, editing isinduced 1007 where transcription of the nuclease and gRNA is induced andthe cells in the culture 1008 are edited and then allowed to recoverfrom editing. Induction of editing in some embodiments comprises raisingthe temperature of the bulk liquid culture to 42° C. to activate the pLpromoter driving transcription of the nuclease, editing gRNA, and donorDNA. Once recovered, the cells are washed, and resuspended in medium1009 and again outgrown so that the cells are in the stationary phase ofgrowth or nearly so. The editing vectors in the cells are then cured1010 by inducing transcription of the curing gRNA that cuts the pUCorigin of replication on the editing vector. Induction of the anti-pUCgRNA is accomplished by first raising the temperature of the culture to42° C. for 2 hours, thereby inducing the pL promoter drivingtranscription of the nuclease, and second by the addition of 2,4diacetylphloroglucinol (DAPG) to induce the pPhIF promoter drivingtranscription of the anti-pUC gRNA (see the vector architectures of theexemplary engine and editing vectors in FIGS. 1C and 1D).

After 2 hours at 42° C., the temperature of the cell culture is loweredto 30° C., and the cells are allowed to recover and grow for 6 additonalhours. The co-expression of the nuclease and the anti-pUC gRNA permitstargeting of the pUC origin of expression on the editing vector. Growingthe cells further performs “passive editing” as described above. At step1011, the cells are washed with medium containing chloramphenicol (againto select for cells comprising the engine vector), and the cells areallowed to recover. At this point, the edited cells can be madeelectrocompetent once more 1012 and be subjected to a second round ofediting.

FIG. 1I depicts an exemplary recursive method 160 c using a solid wallisolation device. The recursive method 160 c—as with the recursivemethods 160 a and 160 b—begins with competent cells 162, for example, E.coli cells that have been previously transformed with an engine vectorexpressing a nucleic acid-guided nuclease such as MAD7 and a selectivemarker, such as a chloramphenicol resistance gene. At step 163, thecompetent cells are transformed with a library of editing vectors, whereeach editing vector in the library of editing vectors comprises anediting cassette with a sequence coding for an editing gRNA and a donorDNA, and each editing vector also comprises an antibiotic resistancegene such as a gene conferring resistance to carbenicillin or otherantibiotic gene different from the antibiotic gene on the engine vector.Following transformation, the cells are allowed to recover 164 in mediumwithout antibiotic, and then at step 165, the cells are diluted, ifnecessary, and loaded onto a solid wall singulation, induction,isolation and normalization device (a SWIIN, described in detail belowin relation to FIGS. 6B-6E) 166 c where the editing process takes place.The cells are loaded into the SWIIN in a Poisson or substantial Poissondistribution (described in detail below) and are grown for at 30° C. forapproximately 8-9 hours. After the initial growth phase, medium exchangeis performed, adding arabinose to the culture medium to induce thelambda Red recombination system encoded by, e.g., the engine vector(see, e.g.,the exemplary vector architectures of FIGS. 1C and 1D).

Once the lambda Red recombination system is induced for, e.g., 30minutes to an hour, the temperature of the SWIIN is increased 42° C. for2 hours, thereby inducing the pL promoters driving transcription of thenuclease on the engine vector and the editing cassette (editing gRNA anddonor DNA) on the editing vector and thus inducing editing. After 2hours at 42° C., the cells are grown at 30° C. for at least 9 morehours.

At step 175, the cells are recovered from the SWIIN and washed to, e.g.,remove the carbenicillin. The cells are then resuspended in mediumcontaining chloramphenicol (continuing to select for the engine vector)and out-grown so that the cells are in late log phase or stationaryphase. The editing vectors in the cells are then cured 168 by inducingtranscription of the curing gRNA that cuts the pUC origin of replicationon the editing vector. Induction of the anti-pUC gRNA is accomplished byfirst raising the temperature of the culture to 42° C. for 2 hours,thereby inducing the pL promoter driving transcription of the nuclease,and second by performing media exchange to medium with of 2,4diacetylphloroglucinol (DAPG) added to induce the pPhIF promoter drivingtranscription of the anti-pUC gRNA (see FIGS. 1C and 1D).

After 2 hours at 42° C., the temperature of the cell culture is loweredto 30° C., and the cells are allowed to recover and grow for 6 hours.The co-expression of the nuclease and the anti-pUC gRNA permitstargeting of the pUC origin of expression on the editing vector. At step169, the cells are washed with medium containing chloramphenicol (againto select for cells comprising the engine vector), and the cells areallowed to recover and are grown to be made electrocompetent. Growingthe cells also performs “passive” curing, where the editing vector is“diluted out” of the growing cell population, as removing from themedium the antibiotic that selects for the editing vector removespressure on cells to retain the editing vector. At this point, theedited cells can be made electrocompetent once more 170 and be subjectedto a second round of editing 171. FIG. 6A depicts and the description ofFIG. 6A presents this method in additional detail.

Automated Cell Editing Instruments and Modules to Perform NucleicAcid-Guided Nuclease Editing including Curing

Automated Cell Editing Instruments

FIG. 2A depicts an exemplary automated multi-module cell processinginstrument 200 to, e.g., perform one of the exemplary recursiveworkflows for targeted gene editing of live yeast cells. The instrument200, for example, may be and preferably is designed as a stand-alonedesktop instrument for use within a laboratory environment. Theinstrument 200 may incorporate a mixture of reusable and disposablecomponents for performing the various integrated processes in conductingautomated genome cleavage and/or editing in cells without humanintervention. Illustrated is a gantry 202, providing an automatedmechanical motion system (actuator) (not shown) that supplies XYZ axismotion control to, e.g., an automated (i.e., robotic) liquid handlingsystem 258 including, e.g., an air displacement pipettor 232 whichallows for cell processing among multiple modules without humanintervention. In some automated multi-module cell processinginstruments, the air displacement pipettor 232 is moved by gantry 202and the various modules and reagent cartridges remain stationary;however, in other embodiments, the liquid handling system 258 may staystationary while the various modules and reagent cartridges are moved.

Also included in the automated multi-module cell processing instrument200 are reagent cartridges 210 comprising reservoirs 212 andtransformation module 230 (e.g., a flow-through electroporation deviceas described in detail in relation to FIGS. 5C-5G and an exemplaryreagent cartridge is described in relation to FIGS. 5A and 5B), as wellas wash reservoirs 206, cell input reservoir 251 and cell outputreservoir 253. The wash reservoirs 206 may be configured to accommodatelarge tubes, for example, wash solutions, or solutions that are usedoften throughout an iterative process. Although two of the reagentcartridges 210 comprise a wash reservoir 206 in FIG. 2A, the washreservoirs instead could be included in a wash cartridge where thereagent and wash cartridges are separate cartridges. In such a case, thereagent cartridge 210 and wash reservoir 206 may be identical except forthe consumables (reagents or other components contained within thevarious inserts) inserted therein.

In some implementations, the reagent cartridges 210 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 200. For example, a user may open andposition each of the reagent cartridges 210 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 200 prior to activating cell processing.Further, each of the reagent cartridges 210 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 2A is the robotic liquid handling system 258including the gantry 202 and air displacement pipettor 232. In someexamples, the robotic handling system 258 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips 215 may be provided in a pipettetransfer tip supply 214 for use with the air displacement pipettor 232.

Inserts or components of the reagent cartridges 210, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 258.For example, the robotic liquid handling system 258 may scan one or moreinserts within each of the reagent cartridges 210 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 210, and a processing system (not shown, but seeelement 237 of FIG. 2B) of the automated multi-module cell editinginstrument 200 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 2A, acell growth module comprises a cell growth vial 218 (described ingreater detail below in relation to FIGS. 3A-3D). Additionally seen isthe TFF module 222 (described above in detail in relation to FIGS.4A-4E). Also illustrated as part of the automated multi-module cellprocessing instrument 200 of FIG. 2A is a singulation module 240 (e.g.,a solid wall isolation, incubation and normalization device (SWIINdevice) is shown here) described herein in relation to FIGS. 6B-6E,served by, e.g., robotic liquid handing system 258 and air displacementpipettor 232. Additionally seen is a selection module 220. Also note theplacement of three heatsinks 255.

FIG. 2B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 200 depicted in FIG. 2A.Cartridge-based source materials (such as in reagent cartridges 210),for example, may be positioned in designated areas on a deck of theinstrument 200 for access by an air displacement pipettor 232 moved bygantry 202 with pipette tips supplied by pipette transfer tip supply214. The deck of the multi-module cell processing instrument 200 mayinclude a protection sink such that contaminants spilling, dripping, oroverflowing from any of the modules of the instrument 200 are containedwithin a lip of the protection sink. Also seen are reagent cartridges210, which are shown disposed with thermal assemblies 211 which cancreate temperature zones appropriate for different regions. Note thatone of the reagent cartridges also comprises a flow-throughelectroporation device 230 (FTEP), served by FTEP interface (e.g.,manifold arm) and actuator 231. Also seen is TFF module 222 withadjacent thermal assembly 225, where the TFF module is served by TFFinterface (e.g., manifold arm) and actuator 233. Thermal assemblies 225,235, and 245 encompass thermal electric devices such as Peltier devices,as well as heatsinks, fans and coolers. The rotating growth vial 218 iswithin a growth module 234, where the growth module is served by twothermal assemblies 235. Selection module is seen at 220. Also seen isthe SWIIN module 240, comprising a SWIIN cartridge (not shown), wherethe SWIIN module also comprises a thermal assembly 245, illumination 243(in this embodiment, backlighting), evaporation and condensation control249, and where the SWIIN module is served by SWIIN interface (e.g.,manifold arm) and actuator 247. Also seen in this view is touch screendisplay 201, display actuator 203, illumination 205 (one on either sideof multi-module cell processing instrument 200), cooling grate 264, andcameras 239 (one illumination device on either side of multi-module cellprocessing instrument 200). Finally, element 237 comprises electronics,such as circuit control boards, high-voltage amplifiers, power supplies,and power entry; as well as pneumatics, such as pumps, valves andsensors.

FIG. 2C illustrates a front perspective view of multi-module cellprocessing instrument 200 for use in as a desktop version of theautomated multi-module cell editing instrument 200. For example, achassis 290 may have a width of about 24-48 inches, a height of about24-48 inches and a depth of about 24-48 inches. Chassis 290 may be andpreferably is designed to hold all modules and disposable supplies usedin automated cell processing and to perform all processes requiredwithout human intervention; that is, chassis 290 is configured toprovide an integrated, stand-alone automated multi-module cellprocessing instrument. As illustrated in FIG. 2C, chassis 290 includestouch screen display 201, cooling grate 264, which allows for air flowvia an internal fan (not shown). The touch screen display providesinformation to a user regarding the processing status of the automatedmulti-module cell editing instrument 200 and accepts inputs from theuser for conducting the cell processing. In this embodiment, the chassis290 is lifted by adjustable feet 270 a, 270 b, 270 c and 270 d (feet 270a-270 c are shown in this FIG. 2C). Adjustable feet 270 a-270 d, forexample, allow for additional air flow beneath the chassis 290.

Inside the chassis 290, in some implementations, will be most or all ofthe components described in relation to FIGS. 2A and 2B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 210 including a flow-through electroporation device (notshown in this FIG. 2C), a rotating growth vial 218 in a cell growthmodule 234 (not shown in this FIG. 2C), a tangential flow filtrationmodule 222 (not shown in this FIG. 2C), a SWIIN module 240 as well asinterfaces and actuators for the various modules (not shown in this FIG.2C). In addition, chassis 290 houses control circuitry, liquid handlingtubes, air pump controls, valves, sensors, thermal assemblies (e.g.,heating and cooling units) and other control mechanisms (not shown inthis FIG. 2C). For examples of multi-module cell editing instruments,see U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242; 10,421,959;10,465,185; 10,519,437; 10,584,333; and 10,584,334 and U.S. Ser. Nos.16/750,369, filed 23 Jan. 2020; 16/822,249, filed 18 Mar. 2020; and16/837,985, filed 1 Apr. 2020, all of which are herein incorporated byreference in their entirety.

The Rotating Cell Growth Module

FIG. 3A shows one embodiment of a rotating growth vial 300 for use withthe cell growth device and in the automated multi-module cell processinginstruments described herein. The rotating growth vial 300 is anoptically-transparent container having an open end 304 for receivingliquid media and cells, a central vial region 306 that defines theprimary container for growing cells, a tapered-to-constricted region 318defining at least one light path 310, a closed end 316, and a driveengagement mechanism 312. The rotating growth vial 300 has a centrallongitudinal axis 320 around which the vial rotates, and the light path310 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 310 is positioned in the lower constricted portion ofthe tapered-to-constricted region 318. Optionally, some embodiments ofthe rotating growth vial 300 have a second light path 308 in the taperedregion of the tapered-to-constricted region 318. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 310 is shorter than the second light path 308 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 308 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 312 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 312 such that the rotating growth vial 300 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 300 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 300 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 400 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 300 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 300 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 304with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 304 mayoptionally include an extended lip 302 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 300may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 300 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 300 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 300 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 300. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 300 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 3B is a perspective view of one embodiment of a cell growth device330. FIG. 3C depicts a cut-away view of the cell growth device 330 fromFIG. 3B. In both figures, the rotating growth vial 300 is seenpositioned inside a main housing 336 with the extended lip 302 of therotating growth vial 300 extending above the main housing 336.Additionally, end housings 352, a lower housing 332 and flanges 334 areindicated in both figures. Flanges 334 are used to attach the cellgrowth device 330 to heating/cooling means or other structure (notshown). FIG. 3C depicts additional detail. In FIG. 3C, upper bearing 342and lower bearing 340 are shown positioned within main housing 336.Upper bearing 342 and lower bearing 340 support the vertical load ofrotating growth vial 300. Lower housing 332 contains the drive motor338. The cell growth device 330 of FIG. 3C comprises two light paths: aprimary light path 344, and a secondary light path 350. Light path 344corresponds to light path 310 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 300, andlight path 350 corresponds to light path 308 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 316. Lightpaths 310 and 308 are not shown in FIG. 3C but may be seen in FIG. 3A.In addition to light paths 344 and 340, there is an emission board 348to illuminate the light path(s), and detector board 346 to detect thelight after the light travels through the cell culture liquid in therotating growth vial 300.

The motor 338 engages with drive mechanism 312 and is used to rotate therotating growth vial 300. In some embodiments, motor 338 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 338 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 336, end housings 352 and lower housing 332 of the cellgrowth device 330 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 300 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 330 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 330 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 330—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 330, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 3D illustrates a cell growth device 330 as part of an assemblycomprising the cell growth device 330 of FIG. 3B coupled to light source390, detector 392, and thermal componets 394. The rotating growth vial300 is inserted into the cell growth device. Components of the lightsource 390 and detector 392 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 332 that houses the motor that rotatesthe rotating growth vial 300 is illustrated, as is one of the flanges334 that secures the cell growth device 330 to the assembly. Also, thethermal componets 394 illustrated are a Peltier device or thermoelectriccooler. In this embodiment, thermal control is accomplished byattachment and electrical integration of the cell growth device 330 tothe thermal componets 394 via the flange 334 on the base of the lowerhousing 332. Thermoelectric coolers are capable of “pumping” heat toeither side of a junction, either cooling a surface or heating a surfacedepending on the direction of current flow. In one embodiment, athermistor is used to measure the temperature of the main housing andthen, through a standard electronic proportional-integral-derivative(PID) controller loop, the rotating growth vial 300 is controlled toapproximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 300 by piercing though the foil seal orfilm. The programmed software of the cell growth device 330 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 300. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 300 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 330 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 330 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 330 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.For additional details regarding rotating growth vials and cell growthdevices see U.S. Pat. Nos. 10,435,662; 10,443,031; 10,590,375 and U.S.Ser. No. 16/780,640, filed 3 Feb. 2020.

The Cell Concentration Module

As described above in relation to the rotating growth vial and cellgrowth module, in order to obtain an adequate number of cells fortransformation or transfection, cells typically are grown to a specificoptical density in medium appropriate for the growth of the cells ofinterest; however, for effective transformation or transfection, it isdesirable to decrease the volume of the cells as well as render thecells competent via buffer or medium exchange. Thus, one sub-componentor module that is desired in cell processing systems for the processeslisted above is a module or component that can grow, perform bufferexchange, and/or concentrate cells and render them competent so thatthey may be transformed or transfected with the nucleic acids needed forengineering or editing the cell's genome.

FIG. 4A shows a retentate member 422 (top), permeate member 420 (middle)and a tangential flow assembly 410 (bottom) comprising the retentatemember 422, membrane 424 (not seen in FIG. 4A), and permeate member 420(also not seen). In FIG. 4A, retentate member 422 comprises a tangentialflow channel 402, which has a serpentine configuration that initiates atone lower corner of retentate member 422—specifically at retentate port428—traverses across and up then down and across retentate member 422,ending in the other lower corner of retentate member 422 at a secondretentate port 428. Also seen on retentate member 422 are energydirectors 491, which circumscribe the region where a membrane or filter(not seen in this FIG. 4A) is seated, as well as interdigitate betweenareas of channel 402. Energy directors 491 in this embodiment mate withand serve to facilitate ultrasonic welding or bonding of retentatemember 422 with permeate/filtrate member 420 via the energy directorcomponent 491 on permeate/filtrate member 420 (at right). Additionally,countersinks 423 can be seen, two on the bottom one at the top middle ofretentate member 422. Countersinks 423 are used to couple and tangentialflow assembly 410 to a reservoir assembly (not seen in this FIG. 4A butsee FIG. 4B).

Permeate/filtrate member 420 is seen in the middle of FIG. 4A andcomprises, in addition to energy director 491, through-holes forretentate ports 428 at each bottom corner (which mate with thethrough-holes for retentate ports 428 at the bottom corners of retentatemember 422), as well as a tangential flow channel 402 and twopermeate/filtrate ports 426 positioned at the top and center of permeatemember 420. The tangential flow channel 402 structure in this embodimenthas a serpentine configuration and an undulating geometry, althoughother geometries may be used. Permeate member 420 also comprisescountersinks 423, coincident with the countersinks 423 on retentatemember 420.

At bottom of FIG. 4A is a tangential flow assembly 410 comprising theretentate member 422 and permeate member 420 seen in this FIG. 4A. Inthis view, retentate member 422 is “on top” of the view, a membrane (notseen in this view of the assembly) would be adjacent and under retentatemember 422 and permeate member 420 (also not seen in this view of theassembly) is adjacent to and beneath the membrane. Again countersinks423 are seen, where the countersinks in the retentate member 422 and thepermeate member 420 are coincident and configured to mate with threadsor mating elements for the countersinks disposed on a reservoir assembly(not seen in FIG. 4A but see FIG. 4B).

A membrane or filter is disposed between the retentate and permeatemembers, where fluids can flow through the membrane but cells cannot andare thus retained in the flow channel disposed in the retentate member.Filters or membranes appropriate for use in the TFF device/module arethose that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 20 μm. Indeed, the poresizes useful in the TFF device/module include filters with sizes from0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm,0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm,0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm,0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm andlarger. The filters may be fabricated from any suitable non-reactivematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glassfiber, or metal substrates as in the case of laser or electrochemicaletching.

The length of the channel structure 402 may vary depending on the typeand volume of the cell culture to be grown and the optical density ofthe cell culture to be concentrated. The length of the channel structuretypically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80mm to 100 mm. The cross-section configuration of the flow channel 402may be round, elliptical, oval, square, rectangular, trapezoidal, orirregular. If square, rectangular, or another shape with generallystraight sides, the cross section may be from about 10 μm to 1000 μmwide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, orfrom 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, orfrom 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400μm to 600 μm high. If the cross section of the flow channel 402 isgenerally round, oval or elliptical, the radius of the channel may befrom about 50 μm to 1000 μm in hydraulic radius, or from 5μm to 800 μmin hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, orfrom 300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500μm in hydraulic radius. Moreover, the volume of the channel in theretentate 422 and permeate 420 members may be different depending on thedepth of the channel in each member.

FIG. 4B shows front perspective (top) and rear perspective (bottom)views of a reservoir assembly 450 configured to be used with thetangential flow assembly 410 seen in FIG. 4A. Seen in the frontperspective view (e.g., “front” being the side of reservoir assembly 450that is coupled to the tangential flow assembly 410 seen in FIG. 4A) areretentate reservoirs 452 on either side of permeate reservoir 454. Alsoseen are permeate ports 426, retentate ports 428, and three threads ormating elements 425 for countersinks 423 (countersinks 423 not seen inthis FIG. 4B). Threads or mating elements 425 for countersinks 423 areconfigured to mate or couple the tangential flow assembly 410 (seen inFIG. 4A) to reservoir assembly 450. Alternatively or in addition,fasteners, sonic welding or heat stakes may be used to mate or couplethe tangential flow assembly 410 to reservoir assembly 450. In additionis seen gasket 445 covering the top of reservoir assembly 450, withpipette tip 405 shown inserted into the left-most retentate reservoir.Gasket 445 is described in detail in relation to FIG. 4E. At left inFIG. 4B is a rear perspective view of reservoir assembly 450, where“rear” is the side of reservoir assembly 450 that is not coupled to thetangential flow assembly. Seen are retentate reservoirs 452, permeatereservoir 454, gasket 445, with pipette tip 405 shown inserted into theright-most retentate reservoir.

The TFF device may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIG. 4C depicts a top-down view of the reservoir assemblies 450 shown inFIG. 4B. FIG. 4D depicts a cover 444 for reservoir assembly 450 shown inFIG. 4B and 4E depicts a gasket 445 that in operation is disposed oncover 444 of reservoir assemblies 450 shown in FIG. 4B. FIG. 4C is atop-down view of reservoir assembly 450, showing the tops of the tworetentate reservoirs 452, one on either side of permeate reservoir 454.Also seen are grooves 432 that will mate with a pneumatic port (notshown), and fluid channels 434 that reside at the bottom of retentatereservoirs 452, which fluidically couple the retentate reservoirs 452with the retentate ports 428 (not shown), via the through-holes for theretentate ports in permeate member 420 and membrane 424 (also notshown). FIG. 4D depicts a cover 444 that is configured to be disposedupon the top of reservoir assembly 450. Cover 444 has round cut-outs atthe top of retentate reservoirs 452 and permeate/filtrate reservoir 454.Again at the bottom of retentate reservoirs 452 fluid channels 434 canbe seen, where fluid channels 434 fluidically couple retentatereservoirs 452 with the retentate ports 428 (not shown). Also shown arethree pneumatic ports 430 for each retentate reservoir 452 andpermeate/filtrate reservoir 454. FIG. 4E depicts a gasket 445 that isconfigured to be disposed upon the cover 444 of reservoir assembly 450.Seen are three fluid transfer ports 442 for each retentate reservoir 452and for permeate/filtrate reservoir 454. Again, three pneumatic ports430, for each retentate reservoir 452 and for permeate/filtratereservoir 454, are shown.

The overall work flow for cell growth comprises loading a cell cultureto be grown into a first retentate reservoir, optionally bubbling air oran appropriate gas through the cell culture, passing or flowing the cellculture through the first retentate port then tangentially through theTFF channel structure while collecting medium or buffer through one orboth of the permeate ports 426, collecting the cell culture through asecond retentate port 428 into a second retentate reservoir, optionallyadding additional or different medium to the cell culture and optionallybubbling air or gas through the cell culture, then repeating theprocess, all while measuring, e.g., the optical density of the cellculture in the retentate reservoirs continuously or at desiredintervals. Measurements of optical densities (OD) at programmed timeintervals are accomplished using a 600 nm Light Emitting Diode (LED)that has been columnated through an optic into the retentatereservoir(s) containing the growing cells. The light continues through acollection optic to the detection system which consists of a (digital)gain-controlled silicone photodiode. Generally, optical density is shownas the absolute value of the logarithm with base 10 of the powertransmission factors of an optical attenuator: OD=−log 10 (Powerout/Power in). Since OD is the measure of optical attenuation—that is,the sum of absorption, scattering, and reflection—the TFF device ODmeasurement records the overall power transmission, so as the cells growand become denser in population, the OD (the loss of signal) increases.The OD system is pre-calibrated against OD standards with these valuesstored in an on-board memory accessible by the measurement program.

In the channel structure, the membrane bifurcating the flow channelsretains the cells on one side of the membrane (the retentate side 422)and allows unwanted medium or buffer to flow across the membrane into afiltrate or permeate side (e.g., permeate member 420) of the device.Bubbling air or other appropriate gas through the cell culture bothaerates and mixes the culture to enhance cell growth. During theprocess, medium that is removed during the flow through the channelstructure is removed through the permeate/filtrate ports 406.Alternatively, cells can be grown in one reservoir with bubbling oragitation without passing the cells through the TFF channel from onereservoir to the other.

The overall work flow for cell concentration using the TFF device/moduleinvolves flowing a cell culture or cell sample tangentially through thechannel structure. As with the cell growth process, the membranebifurcating the flow channels retains the cells on one side of themembrane and allows unwanted medium or buffer to flow across themembrane into a permeate/filtrate side (e.g., permeate member 420) ofthe device. In this process, a fixed volume of cells in medium or bufferis driven through the device until the cell sample is collected into oneof the retentate ports 428, and the medium/buffer that has passedthrough the membrane is collected through one or both of thepermeate/filtrate ports 426. All types of prokaryotic and eukaryoticcells—both adherent and non-adherent cells—can be grown in the TFFdevice. Adherent cells may be grown on beads or other cell scaffoldssuspended in medium that flow through the TFF device.

The medium or buffer used to suspend the cells in the cell concentrationdevice/module may be any suitable medium or buffer for the type of cellsbeing transformed or transfected, such as LB, SOC, TPD, YPG, YPAD, MEM,DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media maybe provided in a reagent cartridge as part of a kit.

In both the cell growth and concentration processes, passing the cellsample through the TFF device and collecting the cells in one of theretentate ports 404 while collecting the medium in one of thepermeate/filtrate ports 406 is considered “one pass” of the cell sample.The transfer between retentate reservoirs “flips” the culture. Theretentate and permeatee ports collecting the cells and medium,respectively, for a given pass reside on the same end of TFFdevice/module with fluidic connections arranged so that there are twodistinct flow layers for the retentate and permeate/filtrate sides, butif the retentate port 404 resides on the retentate member ofdevice/module (that is, the cells are driven through the channel abovethe membrane and the filtrate (medium) passes to the portion of thechannel below the membrane), the permeate/filtrate port 406 will resideon the permeate member of device/module and vice versa (that is, if thecell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). Due to the high pressures used to transfer the cell cultureand fluids through the flow channel of the TFF device, the effect ofgravity is negligible.

At the conclusion of a “pass” in either of the growth and concentrationprocesses, the cell sample is collected by passing through the retentateport 428 and into the retentate reservoir (not shown). To initiateanother “pass”, the cell sample is passed again through the TFF device,this time in a flow direction that is reversed from the first pass. Thecell sample is collected by passing through the retentate port 428 andinto retentate reservoir (not shown) on the opposite end of thedevice/module from the retentate port 428 that was used to collect cellsduring the first pass. Likewise, the medium/buffer that passes throughthe membrane on the second pass is collected through the permeate port426 on the opposite end of the device/module from the permeate port 426that was used to collect the filtrate during the first pass, or throughboth ports. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been grown to a desired optical density, and/orconcentrated to a desired volume, and both permeate ports (i.e., ifthere are more than one) can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellgrowth may (and typically do) take place simultaneously, and bufferexchange and cell concentration may (and typically do) take placesimultaneously. For further information and alternative embodiments onTFFs see, e.g., U.S. Ser. No. 16/798,302, filed 22 Feb. 2020.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments of the present disclosure optionally include a nucleic acidassembly module. The nucleic acid assembly module is configured toaccept and assemble the nucleic acids necessary to facilitate thedesired genome editing events. In general, the term “vector” refers to anucleic acid molecule capable of transporting a desired nucleic acid towhich it has been linked into a cell. Vectors include, but are notlimited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat include one or more free ends, no free ends (e.g., circular);nucleic acid molecules that include DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors” or “editingvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. Additional vectors includefosmids, phagemids, and synthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transcription, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in US Pub. No. 2004/0171156, the contents of whichare herein incorporated by reference in their entirety for all purposes.

Regulatory elements are operably linked to one or more elements of atargetable nuclease system so as to drive transcription, and for somenucleic acid sequences, translation and expression of the one or morecomponents of the targetable nuclease system.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g., GoldenGateassembly, European Patent Application Publication EP 2 395 087 A1), andLigase Cycling Reaction (de Kok, ACS Synth Biol., 3(2):97-106 (2014);Engler, et al., PLoS One, 3(11):e3647 (2008); and U.S. Pat. No.6,143,527). In other embodiments, the nucleic acid assembly techniquesperformed by the disclosed automated multi-module cell editinginstruments are based on overlaps between adjacent parts of the nucleicacids, such as Gibson Assembly®, CPEC, SLIC, Ligase Cycling etc.Additional assembly methods include gap repair in yeast (Bessa, Yeast,29(10):419-23 (2012)), gateway cloning (Ohtsuka, Curr Pharm Biotechnol,10(2):244-51 (2009)); U.S. Pat. Nos. 5,888,732; and 6,277,608), andtopoisomerase-mediated cloning (Udo, PLoS One, 10(9):e0139349 (2015);and U.S. Pat. No. 6,916,632). These and other nucleic acid assemblytechniques are described, e.g., in Sands and Brent, Curr Protoc MolBiol., 113:3.26.1-3.26.20 (2016).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automatedmulti-module cell editing instrument. For example, when PCR is utilizedin the nucleic acid assembly module, the module includes a thermocyclingcapability allowing the temperatures to cycle between denaturation,annealing and extension steps. When single temperature assembly methods(e.g., isothermal assembly methods) are utilized in the nucleic acidassembly module, the module provides the ability to reach and hold atthe temperature that optimizes the specific assembly process beingperformed. These temperatures and the duration for maintaining thesetemperatures can be determined by a preprogrammed set of parametersexecuted by a script, or manually controlled by the user using theprocessing system of the automated multi-module cell editing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction. Certain isothermalassembly methods can combine simultaneously up to 15 nucleic acidfragments based on sequence identity. The assembly method provides, insome embodiments, nucleic acids to be assembled which include anapproximate 20-40 base overlap with adjacent nucleic acid fragments. Thefragments are mixed with a cocktail of three enzymes—an exonuclease, apolymerase, and a ligase-along with buffer components. Because theprocess is isothermal and can be performed in a 1-step or 2-step methodusing a single reaction vessel, isothermal assembly reactions are idealfor use in an automated multi-module cell editing instrument. The 1-stepmethod allows for the assembly of up to five different fragments using asingle step isothermal process. The fragments and the master mix ofenzymes are combined and incubated at 50° C. for up to one hour. For thecreation of more complex constructs with up to fifteen fragments or forincorporating fragments from 100 bp up to 10 kb, typically the 2-step isused, where the 2-step reaction requires two separate additions ofmaster mix; one for the exonuclease and annealing step and a second forthe polymerase and ligation steps.

The Cell Transformation Module

FIGS. 5A and 5B depict the structure and components of an embodiment ofan exemplary reagent cartridge useful in the automated multi-moduleinstrument described therein. In FIG. 5A, reagent cartridge 500comprises a body 502, which has reservoirs 504. One reservoir 504 isshown empty, and two of the reservoirs have individual tubes (not shown)inserted therein, with individual tube covers 505. Additionally shownare rows of reservoirs into which have been inserted co-joined rows oflarge tubes 503 a, and co-joined rows of small tubes 503 b. Theco-joined rows of tubes are presented in a strip, with outer flanges 507that mate on the backside of the outer flange (not shown) with anindentation 509 in the body 502, so as to secure the co-joined rows oftubes (503 a and 503 b) to the reagent cartridge 500. Shown also is abase 511 of reagent cartridge body 502. Note that the reservoirs 504 inbody 502 are shaped generally like the tubes in the co-joined tubes thatare inserted into these reservoirs 504.

FIG. 5B depicts the reagent cartridge 500 in FIG. 5A with a row ofco-joined large tubes 503 a, a row of co-joined small tubes 503 b, andone large tube 560 with a cover 505 removed from (i.e., depicted above)the reservoirs 504 of the reagent cartridge 500. Again, the co-joinedrows of tubes are presented in a strip, with individual large tubes 561shown, and individual small tubes 555 shown. Again, each strip ofco-joined tubes comprises outer flanges 507 that mate on the backside(not shown) of the outer flange with an indentation 509 in the body 502,to secure the co-joined rows of tubes (503 a and 503 b) to the reagentcartridge 500. As in FIG. 5A, reagent cartridge body 502 comprises abase 511. Reagent cartridge 500 may be made from any suitable material,including stainless steel, aluminum, or plastics including polyvinylchloride, cyclic olefin copolymer (COC), polyethylene, polyamide,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),polysulfone, and polyurethane, and co-polymers of these and otherpolymers. Again, if reagent cartridge 500 is disposable, it preferablyis made of plastic. In addition, in many embodiments the material usedto fabricate the cartridge is thermally-conductive, as reagent cartridge500 may contact a thermal device (not shown) that heats or coolsreagents in the reagent reservoirs 504, including reagents in co-joinedtubes. In some embodiments, the thermal device is a Peltier device orthermoelectric cooler.

FIGS. 5C and 5D are top perspective and bottom perspective views,respectively, of an exemplary FTEP device 550 that may be part of (e.g.,a component in) reagent cartridge 500 in FIGS. 5A and 5B or may be astand-alone module; that is, not a part of a reagent cartridge or othermodule. FIG. 5C depicts an FTEP device 550. The FTEP device 550 haswells that define cell sample inlets 552 and cell sample outlets 554.FIG. 5D is a bottom perspective view of the FTEP device 550 of FIG. 5C.An inlet well 552 and an outlet well 554 can be seen in this view. Alsoseen in FIG. 5D are the bottom of an inlet 562 corresponding to well552, the bottom of an outlet 564 corresponding to the outlet well 554,the bottom of a defined flow channel 566 and the bottom of twoelectrodes 568 on either side of flow channel 566. The FTEP devices maycomprise push-pull pneumatic means to allow multi-pass electroporationprocedures; that is, cells to electroporated may be “pulled” from theinlet toward the outlet for one pass of electroporation, then be“pushed” from the outlet end of the FTEP device toward the inlet end topass between the electrodes again for another pass of electroporation.Further, this process may be repeated one to many times. For additionalinformation regarding FTEP devices, see, e.g., U.S. Pat. Nos.10,435,713; 10,443,074; 10,323,258; and 10,508,288. Further, otherembodiments of the reagent cartridge may provide or accommodateelectroporation devices that are not configured as FTEP devices, such asthose described in USSN 16/109,156, filed 22 Aug. 2018. For reagentcartridges useful in the present automated multi-module cell processinginstruments, see, e.g., U.S. Pat. Nos. 10,376,889; 10,406,525;10,576,474; and U.S. Ser. Nos. 16/749,757, filed 22 Jan. 2020; and16/827,222, filed 23 Mar. 2020.

Additional details of the FTEP devices are illustrated in FIGS. 5E-5G.Note that in the FTEP devices in FIGS. 5E-5G the electrodes are placedsuch that a first electrode is placed between an inlet and a narrowedregion of the flow channel, and the second electrode is placed betweenthe narrowed region of the flow channel and an outlet. FIG. 5E shows atop planar view of an FTEP device 550 having an inlet 552 forintroducing a fluid containing cells and exogenous material into FTEPdevice 550 and an outlet 554 for removing the transformed cells from theFTEP following electroporation. The electrodes 568 are introducedthrough channels (not shown) in the device.

FIG. 5F shows a cutaway view from the top of the FTEP device 550, withthe inlet 552, outlet 554, and electrodes 568 positioned with respect toa flow channel 566. FIG. 5G shows a side cutaway view of FTEP device 550with the inlet 552 and inlet channel 572, and outlet 554 and outletchannel 574. The electrodes 568 are positioned in electrode channels 576so that they are in fluid communication with the flow channel 566, butnot directly in the path of the cells traveling through the flow channel566. Note that the first electrode is placed between the inlet and thenarrowed region of the flow channel, and the second electrode is placedbetween the narrowed region of the flow channel and the outlet. Theelectrodes 568 in this aspect of the device are positioned in theelectrode channels 576 which are generally perpendicular to the flowchannel 566 such that the fluid containing the cells and exogenousmaterial flows from the inlet channel 572 through the flow channel 566to the outlet channel 574, and in the process fluid flows into theelectrode channels 576 to be in contact with the electrodes 568. In thisaspect, the inlet channel, outlet channel and electrode channels alloriginate from the same planar side of the device. In certain aspects,however, the electrodes may be introduced from a different planar sideof the FTEP device than the inlet and outlet channels.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining.

The components of the FTEP devices may be manufactured separately andthen assembled, or certain components of the FTEP devices (or even theentire FTEP device except for the electrodes) may be manufactured (e.g.,using 3D printing) or molded (e.g., using injection molding) as a singleentity, with other components added after molding. For example, housingand channels may be manufactured or molded as a single entity, with theelectrodes later added to form the FTEP unit. Alternatively, the FTEPdevice may also be formed in two or more parallel layers, e.g., a layerwith the horizontal channel and filter, a layer with the verticalchannels, and a layer with the inlet and outlet ports, which aremanufactured and/or molded individually and assembled followingmanufacture.

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 508 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

As mentioned, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells to beelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,where the flow channel decreases in width, the flow channel may narrowto between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μmand 2 mm, or between 75 μm and 1 mm. The distance between the electrodesin the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall sizeof the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cmin length, or 4.5 cm to 10 cm in length. The overall width of the FTEPdevice may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cmto 2.5 cm, or from 1 cm to 1.5 cm.

The region of the flow channel that is narrowed is wide enough so thatat least two cells can fit in the narrowed portion side-by-side. Forexample, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute,or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute.The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi,or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device. Flow-through electroporationdevices (either as a stand-alone instrument or as a module in anautomated multi-module system) are described in, e.g., U.S. Pat. Nos.10,435,713; 10,443,074; 10,323,258; and 10,508,288.

Cell Singulation and Enrichment Device

FIG. 6A depicts a solid wall device 6050 and a workflow for singulatingcells in microwells in the solid wall device. At the top left of thefigure (i), there is depicted solid wall device 6050 with microwells6052. A section 6054 of substrate 6050 is shown at (ii), also depictingmicrowells 6052. At (iii), a side cross-section of solid wall device6050 is shown, and microwells 6052 have been loaded, where, in thisembodiment, Poisson or substantial Poisson loading has taken place; thatis, each microwell has few, one or no cells. At (iv), workflow 6040 isillustrated where substrate 6050 having microwells 6052 shows microwells6056 with one cell per microwell, microwells 6057 with no cells in themicrowells, and one microwell 6060 with two cells in the microwell. Instep 6051, the cells in the microwells are allowed to doubleapproximately 2-150 times to form clonal colonies (v), then editing isallowed to occur 6053.

After editing 6053, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 6058),where cells that do not undergo editing thrive (microwells 6059) (vi).All cells are allowed to continue grow to establish colonies andnormalize 6055, where the colonies of edited cells in microwells 6058catch up in size and/or cell number with the cells in microwells 6059that do not undergo editing (vii). Once the cell colonies arenormalized, either pooling 6060 of all cells in the microwells can takeplace, in which case the cells are enriched for edited cells byeliminating the bias from non-editing cells and fitness effects fromediting; alternatively, colony growth in the microwells is monitoredafter editing, and slow growing colonies (e.g., the cells in microwells6058) are identified and selected 6061 (e.g., “cherry picked”) resultingin even greater enrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for yeast cell growth includes LB, SOC, TPD, YPG, YPAD,MEM and DMEM.

A module useful for performing the methods depicted in FIG. 6A is asolid wall isolation, incubation, and normalization (SWIIN) module. FIG.6B depicts an embodiment of a SWIIN module 650 from an exploded topperspective view. In SWIIN module 650 the retentate member is formed onthe bottom of a top of a SWIIN module component and the permeate memberis formed on the top of the bottom of a SWIIN module component.

The SWIIN module 650 in FIG. 6B comprises from the top down, a reservoirgasket or cover 658, a retentate member 604 (where a retentate flowchannel cannot be seen in this FIG. 6B), a perforated member 601 swagedwith a filter (filter not seen in FIG. 6B), a permeate member 608comprising integrated reservoirs (permeate reservoirs 652 and retentatereservoirs 654), and two reservoir seals 662, which seal the bottom ofpermeate reservoirs 652 and retentate reservoirs 654. A permeate channel660 a can be seen disposed on the top of permeate member 608, defined bya raised portion 676 of serpentine channel 660 a, and ultrasonic tabs664 can be seen disposed on the top of permeate member 608 as well. Theperforations that form the wells on perforated member 601 are not seenin this FIG. 6B; however, through-holes 666 to accommodate theultrasonic tabs 664 are seen. In addition, supports 670 are disposed ateither end of SWIIN module 650 to support SWIIN module 650 and toelevate permeate member 608 and retentate member 604 above reservoirs652 and 654 to minimize bubbles or air entering the fluid path from thepermeate reservoir to serpentine channel 660 a or the fluid path fromthe retentate reservoir to serpentine channel 660 b (neither fluid pathis seen in this FIG. 6B). Also seen is a gasket 658, which covers thepermeate and retentate reservoir access apertures 632 a, 632 b, 632 c,and 632 d, as well as pneumatic ports 633 a, 633 b, 633 c and 633 d.

In this FIG. 6B, it can be seen that the serpentine channel 660 a thatis disposed on the top of permeate member 608 traverses permeate member608 for most of the length of permeate member 608 except for the portionof permeate member 608 that comprises permeate reservoirs 652 andretentate reservoirs 654 and for most of the width of permeate member608. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types (e.g., such as formammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm ormore. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

Serpentine channels 660 a and 660 b can have approximately the samevolume or a different volume. For example, each “side” or portion 660 a,660 b of the serpentine channel may have a volume of, e.g., 2 mL, orserpentine channel 660 a of permeate member 608 may have a volume of 2mL, and the serpentine channel 660 b of retentate member 604 may have avolume of, e.g., 3 mL. The volume of fluid in the serpentine channel mayrange from about 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5mL to 40 mL, or from 6 mL to 20 mL (note these volumes apply to a SWIINmodule comprising a, e.g., 50-500K perforation member). The volume ofthe reservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, orfrom 8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of allreservoirs may be the same or the volumes of the reservoirs may differ(e.g., the volume of the permeate reservoirs is greater than that of theretentate reservoirs).

The serpentine channel portions 660 a and 660 b of the permeate member608 and retentate member 604, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness.Embodiments the retentate (and permeate) members may be fabricated fromPMMA (poly(methyl methacrylate) or other materials may be used,including polycarbonate, cyclic olefin co-polymer (COC), glass,polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers. Preferably atleast the retentate member is fabricated from a transparent material sothat the cells can be visualized (see, e.g., FIG. 6E and the descriptionthereof). For example, a video camera may be used to monitor cell growthby, e.g., density change measurements based on an image of an emptywell, with phase contrast, or if, e.g., a chromogenic marker, such as achromogenic protein, is used to add a distinguishable color to thecells. Chromogenic markers such as blitzen blue, dreidel teal, virginiaviolet, vixen purple, prancer purple, tinsel purple, maccabee purple,donner magenta, cupid pink, seraphina pink, scrooge orange, and leororange (the Chromogenic Protein Paintbox, all available from ATUM(Newark, Calif.)) obviate the need to use fluorescence, althoughfluorescent cell markers, fluorescent proteins, and chemiluminescentcell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Automatedcolony pickers may be employed, such as those sold by, e.g., TECAN(Pickolo™ system, Mannedorf, Switzerland); Hudson Inc. (RapidPick™Springfield, N.J.); Molecular Devices (QPix 400™ system, San Jose,Calif.); and Singer Instruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 650 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 650, or by applying atransparent heated lid over at least the serpentine channel portion 660b of the retentate member 604. See, e.g., FIG. 6E and the descriptionthereof infra.

In SWIIN module 650 cells and medium—at a dilution appropriate forPoisson or substantial Poisson distribution of the cells in themicrowells of the perforated member—are flowed into serpentine channel660 b from ports in retentate member 604, and the cells settle in themicrowells while the medium passes through the filter into serpentinechannel 660 a in permeate member 608. The cells are retained in themicrowells of perforated member 601 as the cells cannot travel throughfilter 603. Appropriate medium may be introduced into permeate member608 through permeate ports 611. The medium flows upward through filter603 to nourish the cells in the microwells (perforations) of perforatedmember 601. Additionally, buffer exchange can be effected by cyclingmedium through the retentate and permeate members. In operation, thecells are deposited into the microwells, are grown for an initial, e.g.,2-100 doublings, editing may be induced by, e.g., raising thetemperature of the SWIIN to 42° C. to induce a temperature-induciblepromoter or by removing growth medium from the permeate member andreplacing the growth medium with a medium comprising a chemicalcomponent that induces an inducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module650 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 660 a and thus to filter 603 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 6C is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 6C, it canbe seen that serpentine channel 660 a is disposed on the top of permeatemember 608 is defined by raised portions 676 and traverses permeatemember 608 for most of the length and width of permeate member 608except for the portion of permeate member 608 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 652can be seen). Moving from left to right, reservoir gasket 658 isdisposed upon the integrated reservoir cover 678 (cover not seen in thisFIG. 6C) of retentate member 604. Gasket 658 comprises reservoir accessapertures 632 a, 632 b, 632 c, and 632 d, as well as pneumatic ports 633a, 633 b, 633 c and 633 d. Also at the far left end is support 670.Disposed under permeate reservoir 652 can be seen one of two reservoirseals 662. In addition to the retentate member being in cross section,the perforated member 601 and filter 603 (filter 603 is not seen in thisFIG. 6C) are in cross section. Note that there are a number ofultrasonic tabs 664 disposed at the right end of SWIIN module 650 and onraised portion 676 which defines the channel turns of serpentine channel660 a, including ultrasonic tabs 664 extending through through-holes 666(not seen in this FIG. 6C but see FIG. 6B) of perforated member 601.There is also a support 670 at the end distal reservoirs 652, 654 ofpermeate member 608.

FIG. 6D is a side perspective view of an assembled SWIIIN module 650,including, from right to left, reservoir gasket 658 disposed uponintegrated reservoir cover 678 (not seen) of retentate member 604.Gasket 658 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 658 comprises reservoir access apertures 632 a, 632 b, 632 c, and632 d, as well as pneumatic ports 633 a, 633 b, 633 c and 633 d. Also atthe far-left end is support 670 of permeate member 608. In addition,permeate reservoir 652 can be seen, as well as one reservoir seal 662.At the far-right end is a second support 670.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 660.

FIG. 6E depicts the embodiment of the SWIIN module in FIGS. 6B-6Dfurther comprising a heat management system including a heater and aheated cover. The heated cover facilitates the condensation managementthat is required for imaging. Assembly 698 comprises a SWIIN module 650seen lengthwise in cross section, where one permeate reservoir 652 isseen. Disposed immediately upon SWIIN module 650 is cover 694 anddisposed immediately below SWIIN module 650 is backlight 680, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 682, which is disposed over a heatsink 684. In thisFIG. 6E, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 686 and heat sink 688, as well as twothermoelectric coolers 692, and a controller 690 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells as well as strains of cells thatare, e.g., temperature sensitive, etc., and allows use oftemperature-sensitive promoters. Temperature control allows forprotocols to be adjusted to account for differences in transformationefficiency, cell growth and viability. For more details regarding solidwall isolation incubation and normalization devices see U.S. Pat. Nos.10,533,152; 10,550,363; 10,532,324; 10,625,212; 10,633,626; and10,633,627; and U.S. Ser. Nos. 16/693,630, filed 25 November 2019;16/823,269, filed 18 March 2020; 16/820,292, filed 16 Mar. 2020;16/820,324, filed 16 Mar. 2020; and 16/686,066, filed 15 Nov. 2019.

Use of the Automated Multi-Module Yeast Cell Processing Instrument

FIG. 7 illustrates an embodiment of a multi-module cell processinginstrument. This embodiment depicts an exemplary system that performsrecursive gene editing on a cell population. The cell processinginstrument 700 may include a housing 726, a reservoir for storing cellsto be transformed or transfected 702, and a cell growth module(comprising, e.g., a rotating growth vial) 704. The cells to betransformed are transferred from a reservoir to the cell growth moduleto be cultured until the cells hit a target OD. Once the cells hit thetarget OD, the growth module may cool or freeze the cells for laterprocessing or transfer the cells to a cell concentration module 706where the cells are subjected to buffer exchange and renderedelectrocompetent, and the volume of the cells may be reducedsubstantially. Once the cells have been concentrated to an appropriatevolume, the cells are transferred to electroporation device 708. Inaddition to the reservoir for storing cells 702, the multi-module cellprocessing instrument includes a reservoir for storing the vectorpre-assembled with editing oligonucleotide cassettes 722. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 708, which already contains the cell culturegrown to a target OD. In the electroporation device 708, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery module 710, where thecells recover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 712,where the cells can be stored at, e.g., 4° C. for later processing 714,or the cells may be diluted and transferred to aselection/singulation/growth/induction/editing/normalization (SWIIN)module 720. In the SWIIN 720, the cells are arrayed such that there isan average of one cell per microwell. The arrayed cells may be inselection medium to select for cells that have been transformed ortransfected with the editing vector(s). Once singulated, the cells growthrough 2-50 doublings and establish colonies. Once colonies areestablished, editing is induced by providing conditions (e.g.,temperature, addition of an inducing or repressing chemical) to induceediting. Once editing is initiated and allowed to proceed, the cells areallowed to grow to terminal size (e.g., normalization of the colonies)in the microwells and then the cells are treated to conditions that curethe editing vector from this round. Once cured, the cells can be flushedout of the microwells and pooled, then transferred to the storage (orrecovery) unit 712 or can be transferred back to the growth module 704for another round of editing. In between pooling and transfer to agrowth module, there typically is one or more additional steps, such ascell recovery, medium exchange (rendering the cells electrocompetent),cell concentration (typically concurrently with medium exchange by,e.g., filtration. Note that theselection/singulation/growth/induction/editing/normalization and editingmodules may be the same module, where all processes are performed in,e.g., a solid wall device, or selection and/or dilution may take placein a separate vessel before the cells are transferred to the solid wallsingulation/growth/induction/editing/normalization/editing module (solidwall device). Similarly, the cells may be pooled after normalization,transferred to a separate vessel, and cured in the separate vessel. Asan alternative to singulation in, e.g., a solid wall device, thetransformed cells may be grown in—and editing can be induced in—bulkliquid as described above in relation to FIGS. 1G and 1H above. Once theputatively-edited cells are pooled, they may be subjected to anotherround of editing, beginning with growth, cell concentration andtreatment to render electrocompetent, and transformation by yet anotherdonor nucleic acid in another editing cassette via the electroporationmodule 708.

In electroporation device 708, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument exemplified in FIG. 7 iscontrolled by a processor 724 configured to operate the instrument basedon user input or is controlled by one or more scripts including at leastone script associated with the reagent cartridge. The processor 724 maycontrol the timing, duration, and temperature of various processes, thedispensing of reagents, and other operations of the various modules ofthe instrument 700. For example, a script or the processor may controlthe dispensing of cells, reagents, vectors, and editingoligonucleotides; which editing oligonucleotides are used for cellediting and in what order; the time, temperature and other conditionsused in the recovery and expression module, the wavelength at which ODis read in the cell growth module, the target OD to which the cells aregrown, and the target time at which the cells will reach the target OD.In addition, the processor may be programmed to notify a user (e.g., viaan application) as to the progress of the cells in the automatedmulti-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 7, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is that double-edited cells AB may be combined with and edited byvectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, andABZ; double-edited cells AC may be combined with and edited by vectorsX, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ; anddouble-edited cells AD may be combined with and edited by vectors X, Y,and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.

In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine +editing vector in a singlevector system). “Curing” is the process in which one or more vectorsused in the prior round of editing is eliminated from the transformedcells as described in detail above in relation to FIGS. 1A-1I. Curingcan be accomplished by, e.g., cleaving the vector(s) using a curingplasmid thereby rendering the editing and/or engine vector (or single,combined vector) nonfunctional; diluting the vector(s) in the cellpopulation via cell growth (that is, the more growth cycles the cells gothrough, the fewer daughter cells will retain the editing or enginevector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine +editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine vector.

FIG. 8 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a bulkliquid growth module for induced editing and enrichment for edited cellsas described above in relation to FIGS. 1G and 1H. The cell processinginstrument 800 may include a housing 826, a reservoir of cells to betransformed or transfected 802, and a growth module (a cell growthdevice) 804. The cells to be transformed are transferred from areservoir to the growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing, or the cells may betransferred to a cell concentration module 830 where the cells arerendered electrocompetent and concentrated to a volume optimal for celltransformation. Once concentrated, the cells are then transferred to anelectroporation device 808 (e.g., transformation/transfection module).Exemplary electroporation devices of use in the automated multi-modulecell processing instruments for use in the multi-module cell processinginstrument include flow-through electroporation devices.

In addition to the reservoir for storing the cells, the system 800 mayinclude a reservoir for storing editing cassettes 816 and a reservoirfor storing an expression vector backbone 818. Both the editingoligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module828, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 822 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 816 or 818. Once the processes carried out by thepurification module 822 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 808, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via an optional filtration module (not shown). Inelectroporation device 808, the assembled nucleic acids are introducedinto the cells. Following electroporation, the cells are transferredinto a combined recovery/selection module 830. For examples ofmulti-module cell editing instruments, see U.S. Pat. Nos. 10,253,316;10,329,559; 10,323,242; 10,421,959; 10,465,185; 10,519,437; 10,584,333;10,584,334; and U.S. Ser. Nos. 16/412,195, filed 14 May 2019;16/750,369, 23 Jan. 2020; 16/822,249, filed 18 Mar. 2020; and16/837,985, filed 1 Apr. 2020, both of which are herein incorporated byreference in their entirety.

Following recovery, and, optionally, selection, the cells aretransferred to a growth, induction, and editing module (bulk liquidculture) 840. The cells are allowed to grow until the cells reach thestationary growth phase (or nearly so), then editing is induced byinduction of transcription of one or both of the nuclease and gRNA. Insome embodiments, editing is induced by transcription of one or both ofthe nuclease and the gRNA being under the control of an induciblepromoter. In some embodiments, the inducible promoter is a pL promoterwhere the promoter is activated by a rise in temperature and“deactivated” by lowering the temperature.

The recovery, selection, growth, induction, editing and storage modulesmay all be separate, may be arranged and combined as shown in FIG. 8, ormay be arranged or combined in other configurations. In certainembodiments, recovery and selection are performed in one module, andgrowth, editing, and re-growth are performed in a separate module.Alternatively, recovery, selection, growth, editing, and re-growth areperformed in a single module.

Once the cells are edited and re-grown (e.g., recovered from editing),the cells may be stored, e.g., in a storage module 812, where the cellscan be kept at, e.g., 4° C. until the cells are used in another round ofediting or retrieved 814. The multi-module cell processing instrument iscontrolled by a processor 824 configured to operate the instrument basedon user input, as directed by one or more scripts, or as a combinationof user input or a script. The processor 824 may control the timing,duration, temperature, and operations of the various modules of thesystem 800 and the dispensing of reagents. For example, the processor824 may cool the cells post-transformation until editing is desired,upon which time the temperature may be raised to a temperature conduciveof genome editing and cell growth. The processor may be programmed withstandard protocol parameters from which a user may select, a user mayspecify one or more parameters manually or one or more scriptsassociated with the reagent cartridge may specify one or more operationsand/or reaction parameters. In addition, the processor may notify theuser (e.g., via an application to a smart phone or other device) thatthe cells have reached the target OD as well as update the user as tothe progress of the cells in the various modules in the multi-modulesystem.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Growth in the Cell Growth Module

One embodiment of the cell growth device as described herein was testedagainst a conventional cell shaker shaking a 5 ml tube and an orbitalshaker shaking a 125 ml baffled flask to evaluate cell growth inbacterial and yeast cells. Additionally, growth of a bacterial cellculture and a yeast cell culture was monitored in real time using anembodiment of the cell growth device described herein in relation toFIGS. 3A-3D.

In a first example, 20 ml EC23 cells (E. coli cells) in LB were grown ina 35 ml rotating growth vial with a 2-paddle configuration at 30° C.using the cell growth device as described herein. The rotating growthvial was spun at 600 rpm and oscillated (i.e., the rotation directionwas changed) every 1 second. In parallel, 5 ml EC23 cells in LB weregrown in a 5 ml tube at 30° C. and were shaken at 750 rpm. OD₆₀₀ wasmeasured at intervals using a NanoDrop™ spectrophotometer (Thermo FisherScientific). The results are shown in FIG. 9. The rotating growthvial/cell growth device performed better than the cell shaker in growingthe cells to OD₆₀₀ 2.6 in slightly over 4 hours. Another experiment wasperformed with the same conditions (volumes, cells, oscillation) theonly difference being a 3-paddle rotating growth vial was employed withthe cell growth device, and the results are shown in FIG. 10. Again, therotating growth vial/cell growth device performed better than the cellshaker in growing the cells to OD₆₀₀ 1.9.

Two additional experiments were performed, this time comparing therotating growth vial/cell growth device to a baffled flask and anorbital shaker. In one experiment, 20 ml EC138 cells (E. coli cells) inLB were grown in a 35 ml rotating growth vial with a 4-paddleconfiguration at 30° C. The rotating growth vial was spun at 600 rpm andoscillated (i.e., the rotation direction was changed) every 1 second. Inparallel, 20 ml EC138 cells in LB were grown in a 125 ml baffled flaskat 30° C. using an orbital shaker. OD₆₀₀ was measured at intervals usinga NanoDrop™ spectrophotometer (Thermo Fisher Scientific). The resultsare shown in FIG. 11, demonstrating that the rotating growth vial/cellgrowth device performed as well as the orbital shaker in growing thecells to OD₆₀₀ 1.0. In a second experiment 20 ml EC138 cells (E. colicells) in LB were grown in a 35 ml rotating growth vial with a 2-paddleconfiguration at 30° C. using the cell growth device as describedherein. The rotating growth vial was spun at 600 rpm and oscillated(i.e., the rotation direction was changed) every 1 second. In parallel,20 ml EC138 cells in LB were grown in a 125 ml baffled flask at 30° C.using an orbital shaker. OD₆₀₀ was measured at intervals using aNanoDrop™ spectrophotometer (Thermo Fisher Scientific). The results areshown in FIG. 12, demonstrating that the rotating growth vial/cellgrowth device performed as well—or better—as the orbital shaker ingrowing the cells to OD₆₀₀ 1.2.

In yet another experiment, the rotating growth vial/cell growth devicewas used to measure OD₆₀₀ in real time. FIG. 13 is a graph showing theresults of real time measurement of growth of an EC138 cell culture at30° C. using oscillating rotation and employing a 2-paddle rotatinggrowth vial. Note that OD₆₀₀ 2.6 was reached in 4.4 hours.

In another experiment, the rotating growth vial/cell growth device wasused to measure OD₆₀₀ in real time of yeast s288c cells in YPAD. Thecells were grown at 30° C. using oscillating rotation and employing a2-paddle rotating growth vial. FIG. 14 is a graph showing the results.Note that OD₆₀₀ 6.0 was reached in 14 hours.

Example II: Cell Concentration

The TFF module as described above in relation to FIGS. 4A-4E has beenused successfully to process and perform buffer exchange on both E. coliand yeast cultures. In concentrating an E. coli culture, the followingsteps were performed:

First, a 20 ml culture of E. coli in LB grown to OD 0.5-0.62 was passedthrough the TFF device in one direction, then passed through the TFFdevice in the opposite direction. At this point the cells wereconcentrated to a volume of approximately 5 ml. Next, 50 ml of 10%glycerol was added to the concentrated cells, and the cells were passedthrough the TFF device in one direction, in the opposite direction, andback in the first direction for a total of three passes. Again the cellswere concentrated to a volume of approximately 5 ml. Again, 50 ml of 10%glycerol was added to the 5 ml of cells and the cells were passedthrough the TFF device for three passes. This process was repeated; thatis, again 50 ml 10% glycerol was added to cells concentrated to 5 ml,and the cells were passed three times through the TFF device. At the endof the third pass of the three 50 ml 10% glycerol washes, the cells wereagain concentrated to approximately 5 ml of 10% glycerol. The cells werethen passed in alternating directions through the TFF device three moretimes, wherein the cells were concentrated into a volume ofapproximately 400 μl.

Filtrate conductivity and filter processing time was measured for E.coli with the results shown in FIG. 15A. Filter performance wasquantified by measuring the time and number of filter passes required toobtain a target solution electrical conductivity. Cell retention wasdetermined by comparing the optical density (OD₆₀₀) of the cell cultureboth before and after filtration. Filter health was monitored bymeasuring the transmembrane flow rate during each filter pass. Targetconductivity (˜16 μS/cm) was achieved in approximately 30 minutesutilizing three 50 ml 10% glycerol washes and three passes of the cellsthrough the device for each wash. The volume of the cells was reducedfrom 20 ml to 400 and recovery of approximately 90% of the cells hasbeen achieved.

The same process was repeated with yeast cell cultures. A yeast culturewas initially concentrated to approximately 5 ml using two passesthrough the TFF device in opposite directions. The cells were washedwith 50 ml of 1M sorbitol three times, with three passes through the TFFdevice after each wash. After the third pass of the cells following thelast wash with 1M sorbitol, the cells were passed through the TFF devicetwo times, wherein the yeast cell culture was concentrated toapproximately 525 μl. FIG. 15B presents the filter buffer exchangeperformance for yeast cells determined by measuring filtrateconductivity and filter processing time. Target conductivity (˜10 μS/cm)was achieved in approximately 23 minutes utilizing three 50 ml 1Msorbitol washes and three passes through the TFF device for each wash.The volume of the cells was reduced from 20 ml to 525 μl. Recovery ofapproximately 90% of the cells has been achieved.

Example III: Production and Transformation of Electrocompetent E. coliand S. Cerevisiae

For testing transformation of the FTEP device, electrocompetent E. colicells were created. To create a starter culture, 6 ml volumes of LBchlor-25 (LB with 25 μg/ml chloramphenicol) were transferred to 14 mlculture tubes. A 25 μl aliquot of E. coli was used to inoculate the LBchlor-25 tubes. Following inoculation, the tubes were placed at a 45°angle in the shaking incubator set to 250 RPM and 30° C. for overnightgrowth, between 12-16 hrs. The OD600 value should be between 2.0 and4.0. A 1:100 inoculum volume of the 250 ml LB chlor-25 tubes weretransferred to four sterile 500 ml baffled shake flasks, i.e., 2.5 mlper 250 ml volume shake flask. The flasks were placed in a shakingincubator set to 250 RPM and 30° C. The growth was monitored bymeasuring OD600 every 1 to 2 hr. When the OD600 of the culture wasbetween 0.5-0.6 (approx. 3-4 hrs), the flasks were removed from theincubator. The cells were centrifuged at 4300 RPM, 10 min, 4° C. Thesupernatant was removed, and 100 ml of ice-cold 10% glycerol wastransferred to each sample. The cells were gently resuspended, and thewash procedure performed three times, each time with the cellsresuspended in 10% glycerol. After the fourth centrifugation, the cellresuspension was transferred to a 50 ml conical Falcon tube andadditional ice-cold 10% glycerol added to bring the volume up to 30 ml.The cells were again centrifuged at 4300 RPM, 10 min, 4° C., thesupernatant removed, and the cell pellet resuspended in 10 ml ice-coldglycerol. The cells were aliquoted in 1:100 dilutions of cell suspensionand ice-cold glycerol.

The comparative electroporation experiment was performed to determinethe efficiency of transformation of the electrocompetent E. coli usingthe FTEP device described. The flow rate was controlled with a pressurecontrol system. The suspension of cells with DNA was loaded into theFTEP inlet reservoir. The transformed cells flowed directly from theinlet and inlet channel, through the flow channel, through the outletchannel, and into the outlet containing recovery medium. The cells weretransferred into a tube containing additional recovery medium, placed inan incubator shaker at 30° C. shaking at 250 rpm for 3 hours. The cellswere plated to determine the colony forming units (CFUs) that survivedelectroporation and failed to take up a plasmid and the CFUs thatsurvived electroporation and took up a plasmid. Plates were incubated at30° C.; E. coli colonies were counted after 24 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 16A. In FIG. 16A, the left-most bars hatched /// denotecell input, the bars to the left bars hatched \\\ denote the number ofcells that survived transformation, and the right bars hatched ///denote the number of cells that were actually transformed. The FTEPdevice showed equivalent transformation of electrocompetent E. colicells at various voltages as compared to the NEPAGENE™ electroporator.As can be seen, the transformation survival rate is at least 90% and insome embodiments is at least 95%, 96%, 97%, 98%, or 99%. The recoveryratio (the fraction of introduced cells which are successfullytransformed and recovered) is in certain embodiments at least 0.001 andpreferably between 0.00001 and 0.01. In FIG. 16A the recovery ratio isapproximately 0.0001.

Additionally, a comparison of the NEPAGENE™ ELEPO21 and the FTEP devicewas made for efficiencies of transformation (uptake), cutting, andediting. In FIG. 16B, triplicate experiments were performed where thebars hatched /// denote the number of cells input for transformation,and the bars hatched \\\ denote the number of cells that weretransformed (uptake), the number of cells where the genome of the cellswas cut by a nuclease transcribed and translated from a vectortransformed into the cells (cutting), and the number of cells whereediting was effected (cutting and repair using a nuclease transcribedand translated from a vector transformed into the cells, and using aguide RNA and a donor DNA sequence both of which were transcribed from avector transformed into the cells). Again, it can be seen that the FTEPshowed equivalent transformation, cutting, and editing efficiencies asthe NEPAGENE™ electroporator. The recovery rate in FIG. 16B for the FTEPis greater than 0.001.

For testing transformation of the FTEP device in yeast, S. cerevisiaecells were created using the methods as generally set forth inBergkessel and Guthrie, Methods Enzymol., 529:311-20 (2013). Briefly,YFAP media was inoculated for overnight growth, with 3 ml inoculate toproduce 100 ml of cells. Every 100 ml of culture processed resulted inapproximately 1 ml of competent cells. Cells were incubated at 30° C. ina shaking incubator until they reached an OD600 of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD of 150+/−20.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 17. The device showed better transformation and survivalof electrocompetent S. Cerevisiae at 2.5 kV voltages as compared to theNEPAGENE™ method. Input is total number of cells that were processed.

Example IV: Bulk Liquid Protocol: Induction and Outgrowth

250 mL baffled shake flasks were prepared with 50 mL of SOB+100 μg/mLcarbenicillin and 25 μg/mL chloramphenicol. For a full, deconvolutionexperiment, 3 shake flasks were prepared per transformation. 500 μL ofundiluted culture from each transformation reaction was transferred intothe prepared 250 mL shake flasks. The following temperature settingswere set up on an incubator: 30° C. for 9 hours→42° C. for 2 hours→30°C. for 9 hours. This temperature regime was used to allow for additionalrecovery of the cells from transformation during the first eight hours.The lambda red system was induced one hour prior to induction of thenuclease, where lambda induction was triggered by the addition ofarabinose (2.5 mL of 20% arabinose) to the culture, and the nucleaseinduction was triggered by increasing the temperature of the cultures to42° C. For full deconvolution experiments, arabinose was not added tothe UPTAKE and CUT flasks as those should not express lambda red;further, the UPTAKE flasks were not shifted to 42° C.

After the temperature cycling is complete (˜21 hours), the shake flaskswere removed. For NGS-SinglePlex: serial dilutions of 10⁻⁵ to 10⁻⁻⁷ ofeach culture were prepared with 0.8% NaCl (50 μL of culture into 450 μLof sterile, 0.8% NaCl). Following dilution, 300 μL of each dilution wasplated onto 150 mm LB agar plates with standard concentrations ofchloramphenicol and carbenicillin. The plates were then placed in a 30°C. incubator for overnight growth and were picked for singleplex NGS thefollowing day. For NGS-Amplicon: 250 μL of culture from each shake flaskwas removed and used as the input for a plasmid extraction protocol. TheOD of this culture was measured to select a volume based on the desirednumber of cells to go into the plasmid purification. Optionally, anundiluted volume from each shake flask may be plated to seeenrichment/depletion of cassettes and the plates were scraped thefollowing day and processed.

FIG. 18 is a bar graph showing the various types of edits observed usingconstitutive editing in a liquid culture (approximately 20% editingobserved), standard plating procedure (approximately 76% editingobserved), two replica experiments of induced editing in liquid bulk(approximately 70% and 76% editing observed), and two replicaexperiments of induced editing using the standard plating procedure(approximately 60% and 76% editing observed). Editing clonality was alsomeasured. The editing clonality of the standard plating procedure showedmixed clonality for the 96 wells, with some colonies achieving 100%clonality, most colonies achieving greater than 50% clonality, and anaverage clonality of 70% and 60% for two replicates (data not shown).The editing clonality of the liquid bulk procotol shows that themajority of the cells were either 100% edited, or 0% edited (e.g.,wildtype), with a small number (approximately 8%) between 100% or 0%.The average editing efficiency was similar for these protocols.

Example V: Singulation, Growth and Editing of E. coli in 200K SWIIN

Singleplex automated genomic editing using MAD7 nuclease, a library with94 different edits in a single gene (yagP) and employing a 200Ksingulation device such as those exemplified in FIGS. 6B-6E wassuccessfully performed. The engine vector used comprised MAD7 under thecontrol of the pL inducible promoter, and the editing vector usedcomprised the editing cassette being under the control of the pLinducible promoter, and the λ Red recombineering system under control ofthe pBAD inducible promoter pBAD—with the exception that the editingcassette comprises the 94 yagP gene edits (donor DNAs) and theappropriate corresponding gRNAs. Two SWIIN workflows were compared, andfurther were benchmarked against the standard plating protocol. TheSWIIN protocols differ from one another that in one set of replicates LBmedium containing arabinose was used to distribute the cells in theSWIIN (arabinose was used to induce the λ Red recombineering system(which allows for repair of double-strand breaks in E. coli that arecreated during editing), and in the other set of replicates SOB mediumwithout arabinose was used to distribute the cells in the SWIIN and forinitial growth, with medium exchange performed to replace the SOB mediumwithout arabinose with SOB medium with arabinose. Approximately 70Kcells were loaded into the 200K SWIIN.

In all protocols (standard plating, LB-SWIIN, and SOB-SWIIN), the cellswere allowed to grow at 30° C. for 9 hours and editing was induced byraising the temperature to 42° C. for 2.5 hours, then the temperaturewas returned to 30° C. and the cells were grown overnight. The resultsof this experiment are shown in FIG. 19 and in Table 1 below. Note thatsimilar editing performance was observed with the four replicates of thetwo SWIIN workflows, indicating that the performance of SWIIN platingwith and without arabinose in the initial medium is similar. Editingpercentage in the standard plating protocol was approximately 77%, inbulk liquid was approximately 67%, and for the SWIIN replicates rangedfrom approximately 63% to 71%. Note that the percentage of unique editcassettes divided by the total number of edit cassettes was similar foreach protocol.

TABLE 1 SWIIN SWIIN SWIIN SWIIN SOB then SOB then Standard LB/Ara LB/AraSOB/Ara SOB/Ara Plating Rep. A Rep. B Rep. A Rep. B 40006 edit calls/0.777 0.633 0.719 0.663 0.695 identified wells Unique edit cassettes/0.49 0.49 0.43 0.50 0.51 total edit cassettes

Example VI: Curing

Standard Plating Protocol: Three rounds of recursive editing and curingwere performed. Intended edits introduced a stop codon in three sugargenes (Xy1A-Y194*, LacZ-F593*, and GalK-E249*). These mutations cause aloss of function in the target gene. This loss of function phenotype wasobserved by growing cells on MacConkey medium. The editing rate wasdetermined by calculating the ratio of the number of cells with a lossof function mutation to the number of total cells. E. coli 181 cellscomprising the engine vector depicted in FIG. 1C on left were madecompetent and transformed in a 100 μL volume with the editing vectordepicted in FIG. 1C on right. The cells were allowed to recover for 3hours in 3.0 mL total volume SOB medium. The recovered cells were thendiluted 1:100 in 20 mL SOB medium containing chloramphenicol and theappropriate antibiotic for the editing vector. The cells were thenplated on solid SOB medium containing chloramphenicol, the editingvector antibiotic (e.g., carbenicillin, kanamycin, bleomycin,streptomycin or nourseothricin N-acetyl transferase, and arabinose. Thearabinose induces the pBAD promoter driving transcription of the λ Redrecombinase system.

The cells were grown for 9 hours at 30° C., grown for 2 hours at 42° C.to induce the pL promoter driving transcription of the nuclease andediting gRNA, and then grown for another 9 hours at 30° C. The cellswere scraped from the plate and diluted in SOB medium withchloramphenicol only. The cells were then washed 3× and 8 mL of cellswere suspended in 12 mL SOB+chlor medium and grown to OD=3.0 to assurethe cells were in stationary phase. The temperature of the culture wasincreased to 42° C. for two hours to induce the nuclease and 20 μL DAPGwas added to a final concentration of 25 μM to induce transcription ofthe curing gRNA (e.g., the anti-pUC gRNA). The cells were grown at 30°C. for 6 hours. Following curing, the cells were washed in LB+chlormedium, resuspended in LB+chlor medium and grown again at 30° C. toOD=0.5. The cells were washed three times with 10% glycerol to renderthem electrocompetent and subjected to another round of editing. Theengine vector was maintained throughout the rounds of editing. Thesuccession of editing vectors comprised the same editing vectorarchitecture as shown in at right in FIG. 1C; however, the editinggRNA/donor DNA and the selectable marker changed with each round. Theediting gRNAs and editing vector antibiotic resistance genes used foreach round of editing and curing are listed in Table 2.

TABLE 2 SPP experimental description Round 1 2 3 gRNA type editingediting editing Editing locus LacZ GalK XylA Editing vector Abx Carb NatKan

The results for both editing and curing rates are shown in FIG. 20A. Forediting efficiency, note that after a first round of editing, 99% ofcells had one edit; after a second round of editing a small percentageof cells had zero edits, approximately 95% of cells had one edit,approximately 95% of cells had two edits; and after a third round ofediting, a small percentage of cells had zero edits, approximately 90%of cells had one edit, approximately 80% of cells had two edits, andapproximately 38% of cells had all three edits. Note that these numbersare the fraction of cells that have at least 1, 2, or 3 edits; thus, ifa cell has three edits it counts for each category, that is, it is thecumulative editing rate. Curing efficiency was calculated using thefollowing equation:

$1 - \frac{{CFUcassette} + {engine}}{CFUengine}$

As seen in FIG. 20A, curing efficiency was well over 95% for each round.

Bulk Liquid Protocol: Four rounds of recursive editing and curing wereperformed using sugar editing gRNAs. Intended edits introduced a stopcodon in three sugar genes (XylA-Y194*, LacZ-F593*, and GalK-E249*).These mutations cause a loss of function in the target gene. This lossof function phenotype can be observed by growing cells on MacConkeymedium. The editing rate was determined by calculating the ratio of thenumber of cells with a loss of function mutation to the number of totalcells. The fourth-round edit was YiaW_C183 and rate was determined bysampling colonies and Sanger sequencing 96 colonies at the edit loci. E.coli 181 strain cells comprising the engine vector depicted in FIG. 1Cwere made competent and transformed in a 100 μL volume with the editingvector depicted in FIG. 1C. The cells were allowed to recover for 3hours in 3.0 mL total volume SOB medium. The recovered cells were thendiluted 1:100 in 20 mL SOB medium containing chloramphenicol and theappropriate antibiotic for the editing vector (e.g., carbenicillin,kanamycin, nourseothricin N-acetyl transferase). The cells were thenplated outgrown for 8 hours in SOB medium containing chloramphenicol andthe antibiotic appropriate for the editing vector. Arabinose was addedto the medium for a final concentration of 1%.

The cells were then grown for another hour at 30° C., grown for 2 hoursat 42° C. to induce the pL promoter driving transcription of thenuclease and editing gRNA, then grown for another 9 hours at 30° C. Thecells were then pelleted, washed 3', and resuspended in 20 mL SOB mediumwith chloramphenicol only. 12 mL of additional medium was added to 8 mLof the cells in suspension. Curing was induced (e.g., transcription ofthe nuclease and curing gRNA) by raising the temperature of the cellculture to 42° C. for 2 hours and by adding 20 μL DAPG to a finalconcentration of 25 μM. The cells were then grown at 30° C. for 6 hoursto OD=2.5. Following curing, the cells were washed in LB+chlor medium,resuspended in LB+chlor medium and grown again at 30° C. to OD=0.5, andwashed with 10% glycerol for another round of editing. The editing gRNAsand editing vector antibiotic resistance genes are listed in Table 3,and the results are shown in FIG. 20B. The succession of editing vectorscomprised the same editing vector architecture as shown in at right inFIG. 1C; however, the editing gRNA/donor DNA and the selectable markerchanged with each round.

For editing efficiency, note that after a first round of editing, 100%of cells had zero edits, which was expected since in the first round theediting vector did not comprise an editing gRNA or cellular targetsequence; after a second round of editing approximately 30% of cells hadzero edits, and approximately 70% of cells had one edit; and after athird round of editing, approximately 60% of cells had zero edits,approximately 40% of cells had one edit, approximately 30% of cells hadtwo edits; and after a fourth round of editing, approximately 80% ofcells had zero edits, and approximately 5% of cells had each of one, twoor three edits. Note that these numbers are the fraction of cells thathave at least 1, 2, or 3 edits; thus, if a cell has three edits itcounts for each category, that is, it is the cumulative editing rate.The percentage curing achieved was over 95% after each round and wasover 99% for the first two rounds.

TABLE 3 Bulk Liquid experimental description Round 1 2 3 4 gRNA typeNon-editing editing editing editing Editing locus None XylA GalK LacZEditing vector Abx Carb Kan Nat Carb

SWIIN Protocol: Four rounds of recursive editing and curing wereperformed using sugar editing gRNAs. Intended edits introduced a stopcodon in three sugar genes (Xy1A-Y194*, LacZ-F593*, and GalK-E249*).These mutations caused a loss of function in the target gene. This lossof function phenotype can be observed by growing cells on MacConkeymedium. The editing rate was determined by calculating the ratio of thenumber of cells with a loss of function mutation to the number of totalcells. The fourth-round edit was YiaW_C183 and rate was determined bysampling colonies and Sanger sequencing 96 colonies at the edit loci. E.coli 181 strain cells comprising the engine vector depicted in FIG. 1Cwere made competent and transformed in a 100 μL volume with the editingvector depicted in FIG. 1C. The cells were allowed to recover for 3hours in 2.7 mL SOB medium. The recovered cells were suspended in 10 mLand then loaded into a 200K SWIIN and grown for 8 hours in SOB mediumcontaining chloramphenicol and the antibiotic appropriate for theediting vector. Medium exchange was performed with arabinose being addedto the medium for a final concentration of 1%. The cells were then grownfor another hour at 30° C., grown for 2.5 hours at 42° C. to induce thepL promoter driving transcription of the nuclease and editing gRNA, thengrown for another 9 hours at 30° C. The cells were then recovered fromthe SWIIN, washed 3×, and resuspended in 20 mL SOB medium withchloramphenicol only. 12 mL of additional medium was added to 8 mL ofthe cells in suspension. The cells were grown to OD=3.0 and curing wasinduced (e.g., transcription of the curing gRNA and nuclease) by adding20 μL DAPG to a final concentration of 25 μM and the temperature of theculture was increased to 42° C. for two hours.

Following curing, the cells were washed in LB+chlor medium, resuspendedin LB+chlor medium and grown again at 30° C. to OD=0.5 for another roundof editing. The editing gRNAs and editing vector antibiotic resistancegenes are listed in Table 4, and the results for curing and editingefficiency are shown in FIG. 20C. The succession of editing vectorscomprised the same editing vector architecture as shown in at right inFIG. 1C; however, the editing gRNA/donor DNA and the selectable markerchanged with each round. Editing on the SWIIN for the first round ofediting resulted in approximately 20% of the cells having zero edits and80% of the cells having one edit; for the second round of editing,approximately 20% of cells had zero edits, 20% had one edit, and 80% hadtwo edits; for the third round of editing approximately 6% of the cellshad zero edits, 4% had one edit, 40% had two edits, and 60% had threeedits; finally after the fourth round of editing, approximately 80% ofthe cells had zero edits, 8% had one edit, 6% had two edits, and 2% hadthree edits. Note that these numbers are the fraction of cells that haveat least 1, 2, or 3 edits; thus, if a cell has three edits it counts foreach category, that is, it is the cumulative editing rate. All rounds ofediting had a percentage of curing above 85%, and after rounds one andthree, editing percentage was above 95%.

TABLE 4 SWIIN experimental description Round 1 2 3 4 gRNA type editingediting editing editing Editing locus LacZ GalK XylA yiaW Editing vectorAbx Carb Nat Kan Carb

Example VII: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279; and U.S. Ser. Nos. 16/024,831filed 30 Jun. 2018; 16/024,816 filed 30 Jun. 2018; 16/147,353 filed 28Sep. 2018; 16/147,865 filed 30 Sep. 2018; and 16/147,871 filed 30 Jun.2018.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module), and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example VIII: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “ lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶16.

We claim:
 1. A method for curing cells of choice during recursivenucleic acid-directed nuclease editing comprising: designing andsynthesizing a first set of editing cassettes, wherein the first set ofediting cassettes comprises one or more covalently-linked editing gRNAand donor DNA pairs, wherein each covalently-linked editing gRNA anddonor DNA pair is under the control of a first inducible promoter;assembling the first set of editing cassettes into a vector backbonethereby forming a first set of editing vectors, wherein the vectorbackbone comprises a first selectable marker, a curing gRNA under thecontrol of a second inducible promoter, a nuclease under the control ofa third inducible promoter; and a curing target sequence; making cellsof choice electrocompetent; transforming the cells of choice with thefirst set of editing vectors to produce first transformed cells;selecting for the first transformed cells via the first selectablemarker thereby selecting for first selected cells; inducing editing inthe first selected cells by inducing the first and third induciblepromoters thereby inducing transcription of the one or morecovalently-linked editing gRNA and donor DNA pairs and the nucleaseproducing first edited cells; curing the first set of editing vectors inthe first edited cells by inducing the third and second induciblepromoters thereby inducing transcription of the nuclease and curing gRNAwhich cuts the curing target sequence producing first cured cells;growing the first cured cells; rendering the first cured cellselectrocompetent; and transforming the first cured cells with a secondset of editing vectors to produce second transformed cells, wherein thesecond set of editing vectors comprises editing cassettes each with oneor more covalently-linked editing gRNA and donor DNA pairs under thecontrol of the first inducible promoter; a second selectable marker; acuring gRNA under the control of the second inducible promoter; anuclease under the control of the third inducible promoter; and thecuring target sequence.
 2. The method of claim 1, wherein the firstinducible promoter and the third inducible promoters are the sameinducible promoter.
 3. The method of claim 1, wherein the first andthird inducible promoters are pL promoters and either the first set ofediting vectors comprises a c1857 gene under the control of aconstitutive promoter.
 4. The method of claim 1, wherein the curingtarget sequence is a pUC origin of replication.
 5. The method of claim4, wherein the curing gRNA is an anti-pUC origin gRNA.
 6. The method ofclaim 1, wherein the second inducible promoter is a pPhIF promoter. 7.The method of claim 1, further comprising, after the second transformingstep, the steps of: selecting for the second transformed cells via thesecond selectable marker thereby selecting for second selected cells;inducing editing in the second selected cells by inducing the first andthird inducible promoters thereby inducing transcription of the one ormore covalently-linked editing gRNA and donor DNA pairs and the nucleaseproducing second edited cells; curing the second set of editing vectorsin the second edited cells by inducing the third and second induciblepromoters thereby inducing transcription of the nuclease and curing gRNAwhich cuts the curing target sequence producing second cured cells;growing the second cured cells; rendering the second cured cellselectrocompetent; and transforming the second cured cells with a thirdset of editing vectors to produce third transformed cells, wherein thethird set of editing vectors comprises editing cassettes each with oneor more covalently-linked editing gRNAs and donor DNA pairs under thecontrol of the first inducible promoter; a third selectable marker; acuring gRNA under the control of the second inducible promoter; anuclease under the control of the third inducible promoter; and thecuring target sequence.
 8. The method of claim 7, further comprising,after the third transforming step, the steps of: selecting for the thirdtransformed cells via the third selectable markers thereby selecting forthird selected cells; inducing editing in the third selected cells byinducing the first and third inducible promoters thereby inducingtranscription of the one or more editing gRNA and donor DNA pairs andthe nuclease producing third edited cells; curing the third set ofediting vectors in the third edited cells by inducing the third andsecond inducible promoters thereby inducing transcription of thenuclease and curing gRNA which cuts the curing target sequence producingthird cured cells; growing the third cured cells; rendering the thirdcured cells electrocompetent; and transforming the third cured cellswith a fourth set of editing vectors to produce fourth transformedcells, wherein the fourth set of editing vectors comprises editingcassettes each with one or more covalently-linked editing gRNAs anddonor DNA pairs under the control of the first inducible promoter; afourth selectable marker; a curing gRNA under the control of the secondinducible promoter; a nuclease under the control of the third induciblepromoter; and the curing target sequence.
 9. The method of claim 8,wherein the first, second, third and fourth sets of editing cassetteseach comprises a library of editing gRNA and donor DNA pairs.
 10. Themethod of claim 9, wherein each library of editing vectors comprises atleast 1000 different editing gRNA and donor DNA pairs.
 11. The method ofclaim 1, wherein the first and third inducible promoters are selectedfrom a pL promoter, a pPhIF promoter, and a rhamnose inducible promoter.12. The method of claim 1, further comprising after the inducing stepand the curing step, a step of growing the first edited cells until thefirst edited cells reach a stationary phase of growth.
 13. The method ofclaim 1, further comprising after the inducing step and the curing step,a step of growing the first edited cells until the first edited cellsreach at least 60% of log phase growth.
 14. The method of claim 13,further comprising after the inducing step and the curing step, a stepof growing the first edited cells until the first edited cells reach atleast 75% of log phase growth.
 15. The method of claim 1, wherein thecells of choice are bacterial cells and the vector backbone furthercomprises a λ Red recombineering system.
 16. The method of claim 1,wherein the cells of choice are yeast cells and the vector backbone islinear.
 17. The method of claim 1, wherein the nuclease is MAD7.
 18. Themethod of claim 1, further comprising between the transformation stepand the inducing step, singulating the first transformed cells.
 19. Themethod of claim 18, wherein the cells are singulated in a solid wallisolation incubation and normalization (SWIIN) module.
 20. The method ofclaim 19, wherein the selecting, inducing, growing, and curing steps areperformed in the SWIIN.